Methods for treating pulmonary vasoconstriction and asthma

ABSTRACT

This invention is a method for treating or preventing bronchoconstriction or reversible pulmonary vasoconstriction in a mammal, which method includes: a) causing the mammal to inhale a therapeutically effective amount of gaseous nitric oxide, and b) introducing into the mammal a therapeutically effective amount of phosphodiesterase inhibiting compounds; and an inhaler device (1) containing nitric oxide gas, and a phosphodiesterase inhibiting compound (106).

A 371 of PCT/US95/04/23 Apr. 3, 1995.

BACKGROUND OF THE INVENTION

This invention relates to the treatment of pulmonary vasoconstrictionand to the treatment of asthma. This invention was made in the course ofwork supported by the U.S. Government, which has certain rights in theinvention.

Asthma is a chronic disease characterized by intermittent, reversible,widespread constriction of the airways of the lungs in response to anyof a variety of stimuli which do not affect the normal lung. Estimatesof the prevalence of this disease in the U.S. population range fromthree to six percent.

In attempting to unravel the pathogenesis of asthma, the cellular andbiochemical basis (sic) for three important features of the disease havebeen sought: chronic airway inflammation, reversible airflowobstruction, and bronchial hyperreactivity. Theories have pointedvariously to abnormalities in autonomic nervous system control of airwayfunction, in bronchial smooth muscle contractile properties, or in theintegrity of the epithelial cell lining as features that distinguishasthmatic from normal airways . . . . Evidence suggests that the normalepithelial lining functions as more than a simple barrier: epithelialcells may produce a relaxing factor that actively maintains airwaypatency by causing relaxation of smooth muscle. Epithelial desquamationcould contribute to bronchial hyperreactivity because a lesser amount ofrelaxing factor would be produced. ("Asthma", Ch. 14-II in ScientificAmerican Medicine, Vol. 2; Scientific American, Inc.; 1988, p. 2, 4)

Drugs used to treat asthma fall generally into two categories: thosewhich act mainly as inhibitors of inflammation, such as corticosteroidsand cromolyn sodium, and those which act primarily as relaxants of thetracheobronchial smooth muscle, such as theophylline and itsderivatives, beta-adrenergic agonists, and anticholinergics. Some ofthese bronchodilators may be administered orally, while others aregenerally given by intravenous or subcutaneous injection or byinhalation of the drug in an appropriate form, such as aerosolizedpowder (i.e., delivered in the form of a finely divided solid, suspendedin a gas such as air), or aerosolized droplets (delivered in the form ofa fine mist). Asthma patients typically self-administer bronchodilatordrugs by means of a portable, metered-dose inhaler, employed as neededto quell or prevent intermittent asthma attacks.

Conceptually analogous to the narrowing of the airways of the lung whichoccurs in an asthma attack, vasoconstriction is a reversible narrowingof blood vessels attributable to contraction of the smooth muscle of theblood vessels. Such vasoconstriction can lead to abnormally high bloodpressure (hypertension) in the affected portion of the circulatorysystem.

The mammalian circulatory system consists of two separate systems, thesystemic circuit and the pulmonary circuit, which are pumped in tandemby the left and right sides of the heart, respectively. The pulmonarycirculation transports the blood through the lungs, where it picks upoxygen and releases carbon dioxide by equilibrating with theconcentrations of oxygen and carbon dioxide gas in the alveoli. Theoxygen-rich blood then returns to the left side of the heart, fromwhence it is distributed to all parts of the body via the systemiccirculation.

The systemic circulatory system of an adult human typically has a meansystemic arterial pressure ("SAP") of 80-100 mm Hg, whereas a typicalmean pulmonary arterial pressure ("PAP") is approximately 12-15 mm Hg.Normal pulmonary capillary pressure is about 7-10 mm Hg. Considering theinterstitial fluid colloid osmotic pressure (14 mm Hg) and the plasmacolloid oncotic pressure (28 mm Hg), as well as the interstitial freefluid pressure (1-8 mm Hg), the normal lung has a +1 mm Hg net meanfiltration pressure (Guyton, Textbook of Medical Physiology. 6th Ed.; W.B. Saunders Co., Philadelphia, Pa. (1981), p. 295). This nearly balancedpressure gradient keeps the alveoli of a healthy lung free of fluidwhich otherwise might seep into the lung from the circulatory system.

An elevation of the PAP over normal levels is termed "pulmonaryhypertension." In humans, pulmonary hypertension is said to exist whenthe PAP increases by at least 5 to 10 mm Hg over normal levels; PAPreadings as high as 50 to 100 mm Hg over normal levels have beenreported. When the PAP markedly increases, plasma can escape from thecapillaries into the lung interstitium and alveoli: fluid buildup in thelung (pulmonary edema) can result, with an associated decrease in lungfunction that can in some cases be fatal.

Pulmonary hypertension may either be acute or chronic. Acute pulmonaryhypertension is often a potentially reversible phenomenon generallyattributable to constriction of the smooth muscle of the pulmonary bloodvessels, which may be triggered by such conditions as hypoxia (as inhigh-altitude sickness), acidosis, inflammation, or pulmonary embolism.Chronic pulmonary hypertension is characterized by major structuralchanges in the pulmonary vasculature which result in a decreasedcross-sectional area of the pulmonary blood vessels; this may be causedby, for example, chronic hypoxia, thromboembolism, or unknown causes(idiopathic or primary pulmonary hypertension).

Pulmonary hypertension has been implicated in several life-threateningclinical conditions, such as adult respiratory distress syndrome("ARDS") and persistent pulmonary hypertension of the newborn ("PPHN").Zapol et al., Acute Respiratory Failure, p. 241-273, Marcel Dekker, NewYork (1985); Peckham, J. Ped. 93:1005 (1978). PPHN, a disorder thatprimarily affects full-term infants, is characterized by elevatedpulmonary vascular resistance, pulmonary arterial hypertension, andright-to-left shunting of blood through the patent ductus arteriosus andforamen ovale of the newborn's heart. Mortality rates range from 12-50%.Fox, Pediatrics 59:205 (1977); Dworetz, Pediatrics 84:1 (1989).Pulmonary hypertension may also result in a potentially fatal heartcondition known as "cor pulmonale", or pulmonary heart disease. Fishman,"Pulmonary Diseases and Disorders" 2nd Ed., McGraw-Hill, New York(1988).

Attempts have been made to treat pulmonary hypertension by administeringdrugs with known systemic vasodilatory effects, such as nitroprusside,hydralazine, and calcium channel blockers. Although these drugs may besuccessful in lowering the pulmonary blood pressure, they typicallyexert an indiscriminate effect, decreasing not only pulmonary but alsosystemic blood pressure. A large decrease in the systemic vascularresistance may result in dangerous pooling of the blood in the venouscirculation, peripheral hypotension (shock), right ventricular ischemia,and consequent heart failure. Zapol (1985); Radermacher, Anaesthesiology68:152 (1988); Vlahakes, Circulation 63:87 (1981). For example, whenintravenous nitroprusside was administered to 15 patients for treatmentof acute pulmonary hypertension due to ARDS, mean PAP decreased from29.6 to 24.2 mm Hg and pulmonary vascular resistance (PVR) decreased bya mean of 32%, but mean systemic arterial pressure was reduced from 89.6mm Hg to the unacceptably low level of 70 mm Hg (Zapol et al., 1985).Intravenous nitroprusside was not recommended for clinical treatment ofpulmonary hypertension, since it "markedly impairs pulmonary gasexchange by increasing Q_(VA) /Q_(T) " (the mixing of venous andarterial blood via an abnormal shunt). Radermacher (1988).

Physiological relaxation of blood vessels has been reported to resultfrom the release of a very labile non-prostanoid endothelium-derivedrelaxing factor (EDRF) by endothelial cells lining the blood vessels.EDRF stimulates the enzyme guanylate cyclase within the vascular smoothmuscle, with the resulting increase in cyclic GMP causing relaxation ofthis muscle, and thereby reversing vasoconstriction. Ignarro et al.,Proc. Natl. Acad. Sci. USA 84:9265 (1987) and Palmer et al., Nature327:524 (1987) identified the vascular smooth muscle relaxation factorreleased by the endothelium of arteries and veins as nitric oxide("NO"). NO is also believed to be produced by breakdown of organicnitrates such as nitroprusside and glyceryl trinitrate. Ignarro, Circ.Res. 65:1 (1989); Furchgott, FASEB J. 3:2007 (1989). Higenbottam et al.,Ann. Rev. Rest. Dis. Suppl. 137:107 (1988), measured the vasodilatoryeffects of inhaled NO in seven patients with a chronic condition termedprimary pulmonary hypertension. The average PAP of these patients whenbreathing 40 ppm NO was 56.7 mm Hg, compared to 59.6 mm Hg whenbreathing air without added NO, a difference of 2.9 mm Hg, or about 6%of the difference ("ΔPAP") between the pre-treatment PAP and what wouldbe normal PAP. Higenbottam et al. reported an average 9% reduction inPVR in these patients during inhalation of NO. No corresponding decreasein SAP was observed.

When exposed to oxygen, NO gas is unstable and undergoes spontaneousoxidation to NO₂ and higher oxides of nitrogen. These higher nitrogenoxides are toxic to the lung, and can in high concentrations themselvesproduce pulmonary edema. NO is "the most rapidly binding ligand tohaemoglobin so far discovered." Meyer, Eur. Resp. J. 2:494 (1988). In adilute aqueous solution exposed to oxygen, dissolved NO has a half lifeof less than 10 seconds due to rapid oxidation to inorganic nitrite andnitrate. Ignarro, FASEB J. 3:31 (1989). The Occupational Safety andHealth Administration (OSHA) has set the time-weighted averageinhalation limit for NO at 25 ppm for 10 hours. "NIOSH Recommendationsfor Occupational Safety and Health Standards," Morbidity and MortalityWeekly Report, Vol. 37, No. S-7, p. 21 (1988).

SUMMARY OF THE INVENTION

The invention features methods for the prevention and treatment ofasthma attacks or other forms of bronchoconstriction, of acuterespiratory failure, or of reversible pulmonary vasoconstriction (i.e.,acute pulmonary vasoconstriction or chronic pulmonary vasoconstrictionwhich has a reversible component), in mammals (especially humans), whichmethod involves the steps of (1) identifying (by, for example,traditional diagnostic procedures) a mammal in need of such treatment orprevention; (2) causing the mammal to inhale a therapeutically-effectiveconcentration of gaseous nitric oxide (or a therapeutically-effectiveamount of a nitric oxide-releasing compound); and (3) prior to, duringor immediately after the NO-inhalation step, introducing into the mammala therapeutically-effective amount of a phosphodiesterase inhibitor,preferably an inhibitor (such as Zapinast™) which is selective for(i.e., is most active against) a cyclic GMP-specific phosphodiesterase.With respect to a patient suffering from bronchoconstriction, a"therapeutically effective" amount of gaseous nitric oxide or a nitricoxide-releasing compound is an amount which reduces the patient's airwayresistance by 20% or more, as measured by standard methods of pulmonarymechanics. With respect to a patient suffering from pulmonaryvasoconstriction, a "therapeutically effective" amount of gaseous nitricoxide or a nitric oxide-releasing compound is an amount which can induceany one or more of the following: (1) prevention of the onset ofpulmonary vasoconstriction following an injury (such as aspiration ortrauma) that could be expected to result in pulmonary vasoconstriction;(2) a 20% or more decrease in the patient's ΔPVR (the difference betweenthe patient's elevated PVR and "normal" PVR, with normal PVR assumed tobe below 1 mmHg.min/liter for an adult human, unless found to beotherwise for a given patient); (3) a 20% or greater decrease in thepatient's ΔPAP; (4) in adults with acute or chronic respiratory failure(e.g., due to asthma or pneumonia), an improvement in arterial oxygentensions by at least 10 mm Hg; or (5) in an infant, improvedtranspulmonary O₂ transport, as measured by a 10% or greater increase ofupper body (pre-ductal) arterial O₂ saturation. PVR is computed bysubtracting the pulmonary capillary wedge pressure (PCWP) (or leftatrial pressure when available) from the mean pulmonary artery pressure(PAP), and dividing by the cardiac output (CO). PVR levels as high as6-20 mmHg-min/liter have been observed in cases of severe ARDS (Zapol etal., N. Engl. J. Med. 296:476-480, 1977). A "therapeutically effective"amount of a phosphodiesterase inhibitor is herein defined as an amountwhich can increase the duration (i.e., half-time) of the therapeuticeffect of gaseous NO or a NO-releasing compound by at least 100%. Thehalf-time of the therapeutic effect is the time, following cessation oftreatment with NO (or the NO-releasing compound), it takes for therelevant measurement of function (reflecting vasoconstriction orbronchoconstriction) to return to a value halfway between the baselinevalue and the peak value achieved during such treatment. In preferredembodiments, the observed increase in duration of therapeutic effectattributable to the action of the phosphodiesterase inhibitor is atleast 200%, and may be greater than 300%.

The methods herein disclosed are useful for preventing (if given priorto the onset of symptoms) or reversing acute pulmonary vasoconstriction,such as may result from pneumonia, traumatic injury, aspiration orinhalation injury, fat embolism in the lung, acidosis, inflammation ofthe lung, adult respiratory distress syndrome, acute pulmonary edema,acute mountain sickness, asthma, post cardiac surgery acute pulmonaryhypertension, persistent pulmonary hypertension of the newborn,perinatal aspiration syndrome, hyaline membrane disease, acute pulmonarythromboembolism, heparin-protamine reactions, sepsis, asthma, statusasthmaticus, or hypoxia (including that which may occur during one-lunganesthesia), as well as those cases of chronic pulmonaryvasoconstriction which have a reversible component, such as may resultfrom chronic pulmonary hypertension, bronchopulmonary dysplasia, chronicpulmonary thromboembolism, idiopathic or primary pulmonary hypertension,or chronic hypoxia. Nitric oxide gas is preferably administered to amammal with pulmonary vasoconstriction or asthma in accordance with oneor more of the following:

(a) administration for at least three minutes (more preferably at leastsix minutes);

(b) administration in the absence of tobacco smoke;

(c) the inhaled concentration of nitric oxide is at least 0.001 ppm,more preferably at least 0.01 ppm, still more preferably at least 0.5ppm, and most preferably at least 1 ppm (e.g., 5, 10 or 20 ppm). Theconcentration would preferably not exceed 180 ppm of nitric oxide (suchconcentration being monitored by a technique such as chemiluminescence);

(d) the nitric oxide is inhaled as a mixture including nitric oxide,oxygen (O₂), and nitrogen (N₂) gases, most preferably having an F_(I) O₂(i.e., proportion of O₂ gas, by volume) of 0.21-0.99, the proportion ofO₂ in air being 0.21; and

(e) the concentration of NO₂ is monitored and kept within safe limits(e.g., less than 1 ppm). Inhalation of gaseous nitric oxide represents amajor advance in asthma therapy, since the gas has no particles ordroplets to disperse and transport to the respiratory tract. Gases havelong free-diffusion pathways, bypass obstructions (such as constrictedairways) readily, and dissolve directly in tissue without causingimpaction bronchospasm. The beneficial effect of NO gas on bronchialsmooth muscle tone is observed immediately following inhalation, makingNO a useful first defense against bronchospasm. The effect, however, isshort-lived once NO inhalation is discontinued, so the inventionincludes treatment with a phosphodiesterase inhibitor which prevents thebreakdown of cyclic GMP by endogenous phosphodiesterases, thusprolonging the beneficial effect of NO on smooth muscle.

The phosphodiesterase inhibitor may be introduced into the mammal by anysuitable method, including via an oral, transmucosal, intravenous,intramuscular, subcutaneous, or intraperitoneal route. The inhibitor mayalternatively be inhaled by the mammal, in order to introduce itdirectly into the affected lung. In such a case, the inhibitor isadvantageously formulated as a dry powder or as an aerosolized solution,and may optionally be inhaled in a gas containing gaseous nitric oxide.

Inhaled nitric oxide also provides a convenient means for diagnosing thereversibility of chronic pulmonary vasoconstriction in a mammal (inparticular, a human): the affected mammal is caused to inhale gaseousnitric oxide, and any changes in PAP and cardiac output before andduring NO inhalation are noted. If the PAP decreases upon inhalation ofNo while the cardiac output remains constant or increases, or if theΔPVR decreases by a significant amount (e.g., at least 20%, orpreferably at least 30%), then the mammal's chronic pulmonaryvasoconstriction would have been shown to have a reversible componentpotentially treatable with gaseous NO or with NO-releasing compounds (orwith other types of vasodilators) administered systemically or byinhalation therapy.

Known nitric oxide-releasing compounds (also referred to as nitricoxide-donor or nitric oxide-generating compounds) useful in the methodsand devices of the invention can be divided into three categories: (a)nitroso or nitrosyl compounds (e.g., S-nitroso-N-acetylpenicillamine,S-nitroso-L-cysteine, and nitrosoguanidine) characterized by an--NOmoiety that is spontaneously released or otherwise transferred from thecompound under physiological conditions such as obtain in the lung; (b)compounds in which NO is a ligand on a transition metal complex, and assuch is readily released or transferred from the compound underphysiological conditions (e.g., nitroprusside, NO-ferredoxin, or anNO-heme complex); and (c) nitrogen-containing compounds which aremetabolized by enzymes endogenous to the respiratory and/or vascularsystem to produce the NO radical (e.g., arginine, glyceryl trinitrate,isoamyl nitrite, inorganic nitrite, azide, and hydroxylamine). Suchtypes of nitric oxide-releasing compounds and methods for theirsynthesis are well known in the art (see, for example, the followingpublications, each of which is incorporated by reference herein: Edwardset al., Biochemical Pharmacology 30:2531-2538, 1981; Schmidt andKukovetz, Eur. J. Pharmacol. 122:75-79, 1986; Curran et al., FASEB J.5:2085-2092, 1991; Southern et al., FEBS Lett. 276:42-44, 1990; Garg etal., J. Clin. Invest. 83:1774-1777, 1989; Garg et al., Biochem. Biophys.Res. Commun. 171:474-479, 1990; Boje et al., J. Pharmacol. Exp. ther.253:20-26, 1990; Bruene et al., J. Biol. Chem. 264:8455-8458, 1989; andMcNamara et al., Can. J. Physiol. Pharmacol. 58:1446-1456, 1980). Acompound known or believed to be such an No-releasing compound can bedirectly tested for its efficacy in the method of the invention by theuse of animal models in one of the in vivo assays described below.Alternatively, such a compound may first be screened for its ability tostimulate guanylate cyclase, the enzyme to which NO binds and therebyexerts its biological activity, in an in vitro assay such as isdescribed by Ishii et al., Am. J. Physiol. 261:H598-H603, 1991. Thestability of the compound during storage can be ascertained, forexample, by subjecting the stored compound to serial measurements of UVlight absorption at a wavelength characteristic of the NO-containingcompound (typically 595 nm).

Both the phosphodiesterase inhibitor compound and the nitricoxide-releasing compound selected for use in the method of the inventionmay be administered as a powder (i.e., a finely divided solid, eitherprovided pure or as a mixture with a biologically-compatible carrierpowder, or with one or more additional therapeutic compounds) or as aliquid (i.e., dissolved or suspended in a biologically-compatible liquidcarrier, optionally mixed with one or more additional therapeuticcompounds), and can conveniently be inhaled in aerosolized form(preferably including particles or droplets having a diameter of lessthan 10 μm). Carrier liquids and powders that are suitable forinhalation are commonly used in traditional asthma inhalationtherapeutics, and thus are well known to those who develop suchtherapeutics. The optimal dosage range can be determined by routineprocedures by a pharmacologist of ordinary skill in the art. Forexample, a useful dosage level for SNAP would be from 1 to 500 μmoles(preferably 1-200 μmoles) per inhaled dose, with the number ofinhalations necessary varying with the needs of the patient.

Also within the invention is an inhaler device (preferably sufficientlylightweight to be considered portable, i.e. less than 5 kg, and morepreferably less than 1 kg) suitable for the treatment or prevention ofbronchoconstriction or pulmonary vasoconstriction, which device may beof a design similar to those inhalers currently available for thetreatment of asthma attacks, and which contains a phosphodiesteraseinhibitor and either or both of (a) pressurized nitric oxide gas, and(b) a nitric oxide-releasing compound. Such a device would typicallyinclude a vessel containing pressurized gas containing at least 0.1 ppm(preferably at least 1 ppm, more preferably at least 5 ppm, and mostpreferably at least 20 ppm) nitric oxide; a housing defining a lumen anda chamber containing an inhalable phosphodiesterase inhibitor compound,which chamber is in communication with the lumen; and a mechanism, suchas a release valve operable by depressing the valve, for controllablyreleasing the gas into lumen or the chamber (thereby suspending thepharmaceutically-active agent in the released gas); the lumen beingconfigured to route the released gas (and suspended agent, if any) intothe respiratory system of a patient. The lumen may include a tube, mask,or rebreathing chamber such as those typically found on presentlyavailable inhaler devices. The device may also have a mechanism foroptionally releasing the gas into the lumen in a manner that bypassesthe compound in the chamber, thereby permitting the patient to first betreated with the nitric oxide-containing gas alone, followed ifnecessary by a dose of the phosphodiesterase inhibitor compoundsuspended in nitric oxide-containing gas. The device can optionallyinclude another pharmaceutically-active agent, such as a bronchodilatorcompound in liquid or solid form. Such a compound could be any compoundcurrently known or subsequently discovered to be effective incounteracting bronchoconstriction. Types of drugs known to be useful inthe inhalation treatment of asthma include cromolyn sodium;anticholinergic agents (such as atropine and ipratropium bromide); β₂agonists (such as adrenaline, isoproterenol, ephedrine, salbutamol,terbutaline, orciprenaline, fenoterol, and isoetharine), methylxanthines(such as theophylline); calcium-channel blockers (such as verapamil);and glucocorticoids (such as prednisone, prednisolone, dexamethasone,beclomethasone dipropionate, and beclomethasone valerate), as describedin Ch. 39 of Principles of Medical Pharmacology, Fifth Edition, Kalantand Roschlau, Ed. (B. C. Decker Inc., Philadelphia, 1989), hereinincorporated by reference. The use and dosage of these and othereffective bronchodilator drugs in inhalation therapy are well known topractitioners who routinely treat asthmatic patients.

In addition to or instead of the above-described bronchodilator drugs,the inhaler device of the invention may also contain an No-releasingcompound (such as SNAP, S-nitrosocysteine, nitroprusside,nitrosoguanidine, glyceryl trinitrate, isoamyl nitrite, inorganicnitrite, azide, or hydroxylamine), which would provide a long-lastingbronchodilating effect to complement the immediate effects obtained byinhaling NO gas. NO-releasing compounds could be tested for theirusefulness in treating asthma attacks and/or reversible pulmonaryvasoconstriction by in vitro and in vivo assays well known topractitioners who routinely develop therapies for these conditions.Criteria for selecting a therapeutically-useful NO-donor compound willinclude its stability in storage prior to inhalation and its ability todecompose to release NO at a therapeutically beneficial rate upondeposition in the appropriate part of the respiratory tract. Forexample, S-nitroso-N-acetylpenicillamine ("SNAP") has been shown to bestable in its solid form, but under physiological conditions (such as inthe film of physiological fluid on the surface of the bronchiolar oralveolar lumen), the compound readily decomposes to release NO (Ignarro,Circ. Res., 1989). The nitric-oxide-releasing compound could be providedin powder form, or it could be dissolved or suspended in abiologically-compatible liquid carrier. The device of the inventioncould be a portable inhaler similar to those typically used by personswith asthma, but which contains a pressurized mixture of nitrogen gas(or another inert gas) and nitric oxide gas (instead of or in additionto an inert, liquified propellant such as a fluorocarbon, e.g., freon).Alternatively, the pharmaceutically-active agent optionally included inthe device of the invention may be an antimicrobial agent, or asurfactant suitable for the treatment of hyaline membrane disease.

In another preferred embodiment, the device of the invention wouldinclude

a vessel containing a phosphodiesterase inhibiting compound (e.g., inliquid or solid form) suspended in a liquified propellant;

a housing defining (a) a port to which the vessel is mounted and (b) alumen in communication with the port; and

a mechanism for controllably releasing the propellant from the vesselinto the lumen, thereby releasing the compound from the vessel into thelumen; such lumen being configured to route the compound into therespiratory system of a person.

Alternatively, the device could include

a vessel containing a compressed or liquified propellant gas (optionallyincluding at least 0.1 ppm nitric oxide gas);

a housing defining (a) a chamber containing a phosphodiesteraseinhibiting compound, and (b) a lumen in communication with the chamber;and

a mechanism for controllably releasing the gas from the vessel into thechamber (for example, in preset doses), thereby suspending the compoundin the gas; the lumen being configured to route the compound into therespiratory system of a person. The device would preferably be ametered-dose inhaler similar to one of the many designs currentlyavailable, which would automatically dispense, in a puff intended forinhalation in a single or multiple breaths, a set amount of the NO gasand the phosphodiesterase inhibitor when activated by the patient inneed of treatment. A single device may optionally be designed todeliver, at the discretion of the patient, NO gas (diluted in an inertgas such as N₂), with or without the solid or liquid phosphodiesteraseinhibiting compound and/or other bronchodilating agent. Such a"two-stage" design would permit the patient to reserve use of thelonger-acting solid or liquid bronchodilator substance until his or herairways had been opened by the puff of gaseous NO in N₂, thus cuttingdown on the dosage of the solid or liquid pharmaceutical necessary forlasting benefit. The optimal level of NO and/or No-releasing compound tobe dispensed can be determined by a pharmacologist using methods such asthose set forth herein. It is expected that a useful inhaled dose of NOgas for the treatment of asthma would be at least 0.1 ppm for 1/2 min.,and preferably from 5 to 300 ppm for one min, which could be achieved,for example, by packaging the compressed NO to be released from thenozzle of the inhaler (or into a rebreathing tube or mask) at at least1,000 ppm in a mixture with N₂. Self-administered treatment of pulmonaryvasoconstriction might require a concentration of 1,000 to 30,000 ppm NOin N₂ at the nozzle, to deliver 5 ml into a 500 ml tidal volume, inorder to result in an effective level of 10 to 300 ppm NO in the lungsof the patient.

No gas could also be used to bronchodilate and thereby improve thedistribution of other agents administered by inhalation. Examples ofsuch agents frequently administered by inhalation include antibioticsand other antimicrobials (e.g., pentamidine for treatment of pneumocytispneumonia), and surfactant agents such as are given to infants withhyaline membrane disease.

The invention described herein provides a simple, safe, rapid, andefficacious treatment or preventative therapy for asthma attacks, foracute respiratory failure (e.g., ARDS or pneumonia), and forvasoconstrictive pulmonary hypertension. In one embodiment of theinvention, a portable inhaler equipped with a cartridge of compressed NOand an aerosol container of a phosphodiesterase inhibiting compound inpowder or liquid form could be used to administer inhalation therapy forasthma or for pulmonary vasoconstriction either in a hospital setting orin an emergency field situation. Such an inhaler can be carried, forexample, by a person at risk of developing hypoxia, such as a mountainclimber, or by ski patrol personnel who can administer the inhalationtherapy on an emergency basis to skiers stricken with hypoxic pulmonaryedema. Similar inhalers containing bronchodilating agents are routinelycarried by asthmatic individuals. In another embodiment of theinvention, a cartridge of compressed NO and an aerosol container of aphosphodiesterase inhibitor could be connected to a ventilation circuitand used to treat and stabilize newborn infants with PPHN duringtransport from the hospital where delivery occurred to one with anintensive care unit, or used to treat pneumonia and ARDS by mask therapyor mechanical ventilator in a hospital or emergency room.

When a phosphodiesterase inhibiting compound or an NO-releasing compoundis inhaled in solid or liquid form, the particles or droplets aredeposited throughout the respiratory system, with larger particles ordroplets tending to be deposited near the point of entry (i.e., in themouth or nose) and smaller particles or droplets being carriedprogressively further into the respiratory system before being depositedin the trachea, bronchi, and finally the alveoli. (See, e.g., Hounam &Morgan, "Particle Deposition", Ch. 5 in Respiratory Defense Mechanisms,Part 1, Marcel Dekker, Inc., NY; ed. Brain et al., 1977; p. 125.) Aparticle/droplet diameter of 10 μm or less is recommended for use in themethod of the invention. Where pulmonary vasoconstriction is the targetcondition, particle/droplet size should in general be of a sizedistribution appropriate for deposition in the alveoli (i.e., averagingless than 5 μm, with an ideal size around 1-3 μm), while treatment of anasthma attack, which affects mainly the bronchi, would preferably beaccomplished using an inhaled particle/droplet size of approximately 2-8μm. Determination of the preferred carrier (if any), propellant (whichmay include NO diluted in an inert gas such as N₂), design of theinhaler, and formulation of the phosphodiesterase inhibitor in itscarrier are well within the abilities of those of ordinary skill in theart of devising routine asthma inhalation therapies. The portableinhaler could contain a canister of compressed NO, preferably in aninert carrier gas such as N₂, or any alternative means of providing NOgas. In addition, the inhaler could contain aphosphodiesterase-inhibiting compound either mixed in dry form with apropellant or held in a chamber separate from the propellant, or mixedwith a liquid carrier capable of being nebulized to an appropriatedroplet size, or in any other configuration known to those skilled inportable inhaler technology. A few of the several types of inhalerdesigns that have been developed to date are discussed in, for example,U.S. Pat. Nos. 4,667,668; 4,592,348; 4,534,343; and 4,852,561, each ofwhich patents is herein incorporated by reference. Other inhaler designsare described in the Physicians' Desk Reference, 45th Edition, Edward R.Barnhart, Publisher (1991). Each of these and other aerosol-typeinhalers can be adapted to accommodate the delivery of NO gas and/orNO-releasing compounds. Also useful for delivering an NO-releasingcompound formulated in dry powder form is a non-aerosol-type inhalerdevice such as that developed by Allen & Hanburys, Research TrianglePark, N.C.

Since NO gas which enters the bloodstream is rapidly inactivated bycombination with hemoglobin, the bronchodilatory effects of inhaled NOare limited to the ventilated bronchi and the vasodilatory effects ofinhaled NO are limited to those blood vessels near the site of NOpassage into the blood stream: i.e., pulmonary microvessels. Therefore,an important advantage of both the bronchodilating and the pulmonaryvasodilating methods of the invention is that one can selectivelyprevent or treat bronchospasm and/or pulmonary hypertension withoutproducing a concomitant lowering of the systemic blood pressure topotentially dangerous levels. The invention allows for effectivereversal of pulmonary hypertension without the risk of underperfusion ofvital organs, venous pooling, ischemia, and heart failure that mayaccompany systemic vasodilation. Such isolated pulmonary vasodilation isalso important in treating PPHN in newborn infants, as systemicvasodilation aggravates the undesired mixing of oxygenated andde-oxygenated blood through the ductus arteriosus or the foramen ovaleof newborns. Furthermore, by concomitantly bronchodilating andincreasing blood flow to ventilated alveoli, the methods of theinvention improve oxygen transport in patients with asthma or acuterespiratory failure, providing an added benefit not seen with typicalbronchodilatory therapies.

The invention also advantageously provides a simple, rapid, non-invasivemethod of diagnosing those forms of chronic pulmonary hypertension whichwill be responsive to NO inhalation therapy. These patients may benefitfrom long-term inhalation therapy by the method of the invention, orfrom chronic systemic treatment with NO-producing vasodilatory drugs,such as nitroprusside and glyceryl trinitrate, with calcium channelblockers, or with other types of vasodilators.

Other features and advantages of the invention will be apparent from thefollowing detailed description, experimental information, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the NO dose response curve for lambs withU46619-induced pulmonary vasoconstriction.

FIG. 2 is a graph showing the effects of inhaling various concentrationsof NO mixed with O₂, alternating with periods of breathing 60-70% O₂without added NO, on the PAP of lambs receiving continuous infusions ofU46619.

FIG. 3 is a strip chart recording illustrating the effect of causing alamb with U46619-induced pulmonary vasoconstriction to inhale 80 ppm NOfor 6 minutes.

FIG. 4 is a graph showing the effects of inhaling various concentrationsof NO mixed with O₂, alternating with periods of breathing 60-70% O₂without added NO, on the pulmonary vascular resistance (PVR) of lambsreceiving continuous infusions of U46619.

FIGS. 5A and 5B are a pair of graphs comparing the effect of 180 ppminhaled NO with untreated controls breathing air on the PAP and PVR ofsheep in which a heparin-protamine reaction has induced an elevated PAPand PVR.

FIG. 6 is a strip chart recording comparing treatment with PGI₂ and withNo inhalation in an adult human with severe ARDS.

FIG. 7 is a representation of the apparatus and conditions used todeliver NO gas to the lungs of guinea pigs in the course of experimentson bronchodilation, and a summary of the chemiluminescence datacollected at each of three sites in the apparatus.

FIG. 8 is a graph illustrating the effects on nine normal (i.e.,non-bronchoconstricted) guinea pig lungs of inhaling 300 ppm NO gas.

FIG. 9 is a graph illustrating the effects on lung resistance observedin nine experimentally bronchoconstricted guinea pigs during treatmentwith various concentrations of NO gas.

FIG. 10 is a graph comparing lung resistance upon treatment of eightexperimentally bronchoconstricted guinea pigs with variousconcentrations of NO gas.

FIGS. 11 and 12 are graphs illustrating the dose-response curve observedwhen nine experimentally bronchoconstricted guinea pigs were treatedwith various concentrations of NO gas, with response measured as lungresistance (FIG. 11) or as a percentage of the maximal lung resistanceobserved (FIG. 12).

FIG. 13 is a graph illustrating the effects on eightexperimentally-bronchoconstricted guinea pig lungs of long-term (onehour) inhalation of 100 ppm NO, or of methacholine alone.

FIG. 14 is a graph illustrating the additive effects of inhaling bothterbutaline and NO on lung resistance in threeexperimentally-bronchoconstricted guinea pigs.

FIG. 15 is a graph illustrating the additive effects of inhaling bothterbutaline and NO on lung compliance in threeexperimentally-bronchoconstricted guinea pigs.

FIG. 16 is a graph illustrating the changes in lung resistance observedin five experimentally-bronchoconstricted guinea pigs inhaling nebulizedS-nitroso-N-acetylpenicillamine (SNAP).

FIG. 17 is a cross-sectional view of one embodiment of the inhalerdevice of the invention.

FIG. 18 is a cross-sectional view of a second embodiment of the inhalerdevice of the invention.

FIG. 19A is a graph demonstrating the influence of continuous i.v.infusion of Zaprinast (0.1 mg-kg⁻¹ min⁻¹) on magnitude of peak decreasesof mean pulmonary arterial pressure in response to NO inhalation duringpulmonary hypertension induced by U46619 infusion. Values are means ±SE.

FIG. 19B is a graph showing the influence of continuous i.v. infusion ofZaprinast (0.1 mg-kg⁻¹ min⁻¹) on half-times of the vasodilating effectin response to NO inhalation during pulmonary hypertension induced byU46619 infusion. Values are means ±SE. *Significantly different fromcontrol.

FIG. 20 is a graph showing the influence of Zaprinast on plasma cyclicGMP levels at baseline condition (baseline), after Zaprinast loadingdose of 2 mg-kg⁻¹ (Zaprinast), on baseline pulmonary hypertension(U46619), and during 6 minutes' NO inhalation. Values are means ±SE.**Significantly different from control. *Significantly different fromits baseline.

FIG. 21 is a bar graph illustrating the influence of Zaprinast on mixedvenous-aortic difference of plasma cGMP concentration during baselineconditions (baseline), stable pulmonary hypertension induced by U46619(U46619), and during incremental concentrations of NO inhaled. Valuesare means of data from 2 animals.

FIG. 22 is a graph illustrating the effect on mean PAP of intermittentNO inhalation during pulmonary hypertension induced by U46619 in anawake lamb. Nitric oxide (40 ppm) was inhaled for 4-minute periods withand without concomitant infusion of Zaprinast. With Zaprinast, asubsequent 4 minute exposure was repeated each time the ΔPAP wasdecreased by 50 percent.

DETAILED DESCRIPTION

NO/Phosphodiesterase Inhibitor Therapy for Pulmonary Vasoconstriction

The invention provides a simple, rapid, selective, and efficaciousmethod of treating or preventing both acute and certain forms of chronicpulmonary hypertension, without concomitantly lowering the systemicblood pressure of the patient. Pulmonary hypertension is a widespreadclinical manifestation, afflicting diverse groups of patients. Use ofinhaled No combined with phosphodiesterase inhibitor (PDE inhibitor)treatment is currently envisioned for, but not limited to, patientsafflicted with or at risk of developing the following: ARDS, pneumonia,asthma, acute pulmonary edema, acute or chronic hypoxia, alveolarhypoventilation states, high altitude pulmonary edema ("mountainsickness"), PPHN, hyaline membrane disease, acidosis, idiopathicpulmonary hypertension, sepsis, pulmonary thromboembolism, cor pulmonalesecondary to pulmonary hypertension, perinatal aspiration syndrome, andacute pulmonary vasoconstriction in response to protamine reversal ofheparin anticoagulation ("heparin-protamine reaction").

Method for administration

Compressed NO gas may be obtained from a commercial supplier such as AirProducts and Chemicals, Inc. (Allentown, Pa.) or Airco (Murray Hill,N.J.), typically as a mixture of 200-800 ppm NO in pure N₂ gas. It isvital that the NO be obtained and stored as a mixture free of anycontaminating O₂ or higher oxides of nitrogen, as such higher oxides ofnitrogen (which can form by reaction of O₂ with NO) are potentiallyharmful to lung tissues. If desired, purity of the NO may bedemonstrated with chemiluminescence analysis, using known methods, priorto administration to the patient. The NO--N₂ mixture may be blended withair or O₂ through, for example, calibrated rotameters which havepreviously been validated with a spirometer. The final concentration ofNO in the breathing mixture may be verified with a chemical orchemiluminescence technique well known to those in the field (e.g.,Fontijin et al., Anal. Chem. 42:575-579, 1970). Any impurities such asNO₂ can be scrubbed by exposure to NaOH solutions, baralyme, orsodalime. As an additional control, the F_(i) O₂ of the final gasmixture may also be assessed. If desired, the ventilator may have a gasscavenger added to the expiratory outlet to ensure that significantamounts of NO will not escape into the adjacent environment.

In a hospital or emergency field situation, administration of NO gascould be accomplished, for example, by attaching a tank of compressed NOgas in N₂, and a second tank of oxygen or an oxygen/N₂ mixture, to aninhaler designed to mix two sources; by controlling the flow of gas fromeach source, the concentration of NO inhaled by the patient can bemaintained at an optimal level.

NO may be administered to mammals suspected of having acute pulmonaryvasoconstriction, at a concentration of from 0.001 ppm to 40 ppm in air,pure oxygen, or another suitable gas or gas mixture, for as long asneeded. The concentration can be increased to 80 to 180 ppm for shortperiods of time: e.g., 5 min at 180 ppm NO, when an immediate dramaticeffect is desired. Concomitant treatment with a PDE inhibitor decreasesthe total dosage of NO required to produce a satisfactory level ofpulmonary vasodilation for an adequate length of time.

Phosphodiesterase (PDE) Inhibitors

In preferred embodiments of the invention, a therapeutically effectiveamount of a PDE inhibitor is administered prior to, during, orimmediately after NO inhalation. Preferably, the PDE inhibitorselectively inhibits the hydrolysis of cGMP, with minimal effects on thebreakdown of cAMP in smooth muscle cells. The PDE inhibitor may beintroduced into the mammal by any suitable method, including via anoral, transmucosal, intravenous, intramuscular, subcutaneous, orintraperitoneal route. Alternatively, the PDE inhibitor may be inhaledby the mammal. For inhalation, the PDE inhibitor is advantageouslyformulated as a dry powder or an aerosolized solution having a particleor droplet size of less than 10 μm, for optimal deposition in thealveoli. Optionally, the PDE inhibitor can be inhaled in a gascontaining NO.

A preferred PDE inhibitor is Zaprinast™ (M&B 22948;2-o-propoxyphenyl-8-azapurin-6-one; Rhone-Poulenc Rorer, Dagenham Essex,UK). Examples of other PDE inhibitors that may be used in the practiceof the present invention are:

WIN 58237 (1-cyclopentyl-3-methyl-6-(4-pyridyl)pyrazolo3,4-d!pyrimidin-4-(5H)-one), see, e.g., Silver et al., J. Pharmacol.Exp. Ther. 271:1143 (1994);

SCH 48936((+)-6a,7,8,9,9a,10,11,11a-octahydro-2,5-dimethyl-3H-pentalen(6a,1,4,5)imidazo2,1-b!purin-4(5H)-one), see, e.g., Chatterjee et al., Circulation90:I627 (abstract No. 3375) (1994);

KT2-734 (2-phenyl-8-ethoxycycloheptimidazole), see, e.g., Satake et al.,Eur. J. Pharmacol. 251:1 (1994); and E4021 (sodium 1-6-chloro-4-(3,4-methylenedioxybenzyl)-aminoquinazolin-2-y!piperidine-4-carboxylate sesquihydrate), see, e.g., Saeki et al., J. Pharmacol. Exp.Ther. 272:825 (1995).

When using Zaprinast™ according to this invention, the preferred routeof administration is intravenous or oral. The suitable dose range forZaprinast™ or other PDE inhibitors may be determined by one of ordinaryskill in the art.

Assessment of Pulmonary vascular pressure and flow

Pulmonary artery pressure is most accurately monitored with aflow-directed pulmonary artery (PA) catheter, placed percutaneously viaa vein of a patient under local anaesthesia; PA flow is usually measuredusing thermaldilution via such a PA catheter. Alternative methods existfor indirect, non-invasive monitoring: e.g., cardiac ultrasound,monitoring of systolic time intervals, and range-gated dopplertechniques. These alternative methods of monitoring may be superiorwhenever catheterization is impracticable, such as in emergencysituations, in patients who are not good candidates for catheterization,or in on-going treatments or established protocols.

Pharmacological effect of nitric oxide

It is likely that inhaled No acts by diffusing into the vascular spaceadjacent to the alveoli and causing relaxation of pulmonary vascularsmooth muscle, thus permitting an increase in pulmonary blood flow andgas exchange. Preliminary evidence obtained in five humans with severeacute respiratory failure demonstrates that NO (approximately 20 ppm)inhaled during mechanical ventilation for periods up to one monthreduces both pulmonary arterial pressure and Q_(VA) /Q_(T) (theright-to-left shunt: a measure of pulmonary oxygen transportinefficiency), thereby producing a marked increase of the patients'blood oxygen levels. This suggests that NO vasodilation occurs only inventilated alveoli and not in non-ventilated or collapsed alveoli, inmarked contrast to results observed following intravenously administeredvasodilators such as nitroprusside. By localizing delivery of NO in agaseous form directly to the lungs, the dissolved NO can immediatelyexert its pharmacological effect on target vascular smooth muscle, priorto inactivation of the NO by binding to hemoglobin. At the same time,the rapid binding of NO to hemoglobin ensures that any vasodilatoryaction of inhaled NO is solely a local or selective effect in the bloodvessels of the lung, with no concomitant vasodilation downstream in thesystemic circulation.

Diagnosis and treatment of chronic pulmonary hypertension

Chronic pulmonary hypertension is characterized by the obstruction orstructural narrowing of blood vessels in the lungs. To the extent thatthe chronic condition of a particular patient is caused or aggravated byspastic constriction of pulmonary vascular smooth muscle orbronchoconstriction, it may be at least partially ameliorated byinhalation of NO: such cases susceptible to treatment with NO, andpotentially with systemic vasodilators, are readily identified by theirresponse to a brief NO inhalation test (e.g., six minutes inhaling 80ppm NO alternating with six minutes inhaling air without added NO,repeated for two to four cycles), while measuring PAP, PCWP, and cardiacoutput. Responsive cases (e.g., those in which the PVR is reduced by 20%or more) can then be treated either with portable NO inhalation therapy,with inhalation of No-releasing compounds in solid or liquid form, orwith NO-releasing systemic vasodilatory drugs such as glyceryltrinitrate or other non-specific systemic dilators (e.g., calciumchannel blockers).

NO-releasing compound inhalation therapy for pulmonary vasoconstriction

The finding that inhalation of gaseous NO can effectively reversecertain forms of pulmonary vasoconstriction suggests yet another mode ofinhalation therapy for pulmonary vasoconstriction, wherein anNO-releasing compound, rather than gaseous NO, is inhaled. This methodwill provide a longer-lasting beneficial effect than briefly inhalinggaseous NO, as the deposited NO-releasing compound would slowly releaseNO over a relatively long period of time. Formulation and dosage of aselected NO-releasing compound can be determined without undueexperimentation by one of ordinary skill in the art. As one example, atypical single inhaled dose of an NO-releasing compound such asS-nitroso-N-acetylpenicillamine (SNAP) or S-nitrosocysteine in drypowder form could range from 60 to 650 μg of the active compound (NO)per kg bodyweight, for approximately an hour of dilation. In sheep withexperimentally-elevated PA pressure, inhalation of SNAP at 1.3 mg/kgproduced a prolonged reduction in PA pressure.

Inhalation therapy for asthma

Like pulmonary vasoconstriction, spastic constriction of the airwayssuch as occurs in asthma attacks can be reversed by inhalation of eithergaseous NO or an NO-releasing compound in solid or liquid form. GaseousNO would have the advantage of rapid diffusion without particles, andwould also vasodilate the bronchodilated region, thereby improvingarterial oxygen tensions. Concomitant treatment with a PDE inhibitor(delivered by inhalation or by any other acceptable route) increases thelength of time that a given dose of NO is clinically effective.Administration would be as described above, and would typically beinitiated upon the onset of an attack or when an attack is thought to beimminent. If chronic bronchodilation of a given patient is needed, thepatient's entire ambient atmosphere could be charged with NO gas at alow dose (at a high gas turnover rate), such as with a mask or tent.

Inhalation devices

The inhalation therapy of the invention is preferably administered bythe use of one of the inhalation devices of the invention. One of suchdevices 10 is illustrated in cross-section in FIG. 17, which shows ahousing 14 defining a chamber 20 in communication with a lumen 16; avessel 12 containing pressurized gas having at least 1 ppm nitric oxidedissolved in a liquified propellant or compressed inert gas whichcontains a suspension of a solid or liquid PDE inhibitor, which vessel12 is slidably mounted in the chamber 20; a pressure-activated valvemechanism 18 for controllably releasing the pressurized contents of thevessel 12 into the lumen 16; and, constituting one end of the lumen 16,a rebreathing chamber 22 having one-way valves 24 through which air 28can enter the rebreathing chamber 22, but through which the therapeuticgas cannot escape. A patient utilizes the device by pushing the upperend 26 of the vessel 12 which protrudes from the housing 14, therebysliding the vessel 12 down into the chamber 20 and depressing the valvemechanism 18. This causes the pressurized contents of the vessel 12 tobe released into the lumen 16 and the rebreathing chamber 22. Thepatient then inhales a portion of the contents of the rebreathingchamber 22, drawing air 28 through the one-way valve 24 into therebreathing chamber 22 to replace the portion of the contents inhaled bythe patient. A single dose of the therapeutic agent released from thevessel 12 into the rebreathing chamber 22 may take several breaths to besufficiently inhaled by the patient. The total weight of this devicewould be less than 200 grams, so that it is readily portable.

In another preferred embodiment 100, illustrated in FIG. 18, the housing102 defines (a) a first chamber 104 containing an inhalable PDEinhibiting compound 106 and (b) a lumen 108 in communication with thefirst chamber 104. A vessel 110 containing pressurized gas or liquifiedpropellant comprising at least 0.1 ppm nitric oxide is slidably mountedin a second chamber 112 of the housing 102, such that pressure appliedto the top of the vessel 114 causes a pressure-release valve located atthe bottom of the vessel 116 to be depressed against the wall of thehousing 102, thereby opening the valve and releasing a portion of thepressurized contents of the vessel 110 into the first chamber 104. Thepressurized gases so released mix with and suspend as an aerosolizedmist the compound 106 in the first chamber 104. This mist is theninhaled by the patient through the open mouthpiece end 118 of the lumen108. At the option of the patient, tab 120 on spring-loaded hinge 122may be manually depressed by the patient prior to and during the openingof the pressure release valve 116; this acts to temporarily close offthe first chamber 104 from the path of the released pressurized gases,which then escape directly into the lumen 108, bypassing the firstchamber 104 in which is located the PDE inhibiting compound 106. Byfirst inhaling the nitric oxide-containing gas without the compound 106suspended therein, the patient's airways are sufficiently opened tomaximize the potential benefits of subsequently inhaling the PDEinhibiting compound 106, so the patient then releases tab 120, againpushes down on the top of the vessel 114 to open valve 116, and inhalesfrom the open end mouthpiece 118 of lumen 108 the compound 106 suspendedin the pressurized gases so released.

Experimental Information

The applicants submit the following experimental animal and human dataand approved protocol for human studies as examples in support of theapplication.

1. PULMONARY VASODILATION

A. Administration of gaseous nitric oxide to lambs

i. Methods

Surgical preparation of the animal model

Eight Suffolk lambs weighing 25-35 kg underwent a sterile thoracotomy inorder to place a left atrial line, tracheostomy and femoral artery lineunder general endotracheal anesthesia with halothane/oxygen three daysbefore study. After three days of recovery the lambs underwent sterileplacement of a 7 French thermal dilution pulmonary artery monitoringcatheter under local anesthesia.

study conditions

Awake unanesthetized lambs were studied in order to avoid generalanesthesia which can blunt hypoxic vasoconstriction. Lambs were placedin a Babraham cage and allowed to drink and eat ad lib. Two studies wereperformed 2 days apart on each of six lambs. After the study the lambswere sacrificed with an overdose of barbiturate and their lungs werefixed, stained and examined by light microscopy for pathologicalchanges.

Administration of NO to lambs with pulmonary vasoconstriction inducedwith U46619

On the first study day lambs breathing 60-70% oxygen were given aninfusion of a potent pulmonary vasoconstrictor, the stable endoperoxideanalog (5Z, 9α, 13E, 15S)-11,9-(Epoxymethano)prosta-5,13-dien-1-oic acid(U46619, The Upjohn Company, Kalamazoo, Mich.) of thromboxane at a rateof 0.4-0.8 μg/kg/min. The tracheostomy was connected to anon-rebreathing circuit consisting of a 5 liter reservoir bag and oneway valves to isolate inspired from expired gas. Expired gas wasscavenged and discarded. The inspired gas was a precise mixture ofoxygen and nitrogen immediately diluted with NO to produce the correctinspired concentration. Using volumetrically calibrated flowmeters,varying quantities of NO were mixed with N₂ to obtain the desiredinspired NO concentration at an inspired oxygen concentration (F_(i) O₂)of 0.6-0.7. The reservoir bag was emptied after each level of NOinhalation. The residence half time of NO in the gas reservoir was 15seconds or less to minimize conversion to NO₂. NO was obtained from AirProducts and Chemicals, Inc., Allentown, Pa. as a mixture of 235 ppm NOin pure N₂. Chemiluminescence analysis demonstrated less than 12 ppm NO₂in this mixture. Fontijin, Anal. Chem. 27:1903 (1981).

A pulmonary vasodilator dose response curve plotting changes in PAP as afunction of inhaled NO concentration during U46619 infusion was producedfor eight lambs breathing a series of increasing NO/O₂ mixtures of 5,10, 20, 40, and 80 ppm NO for six minutes (FIG. 1). Each level of Noexposure was followed by six minutes of breathing the oxygen mixturewithout NO (FIG. 2). A second exposure to NO was examined for similarperiods. Subsequently, a control period breathing the oxygen mixture wasstudied six minutes after ceasing U46619 infusion. At each three and sixminute time period after administering or discontinuing NO during thestudy, we measured mean and phasic pulmonary artery pressure (PAP), leftatrial pressure (LAP), systemic arterial pressure (SAP) and centralvenous pressure (CVP). All pressures were recorded on a Hewlett Packardmulti-channel strip chart recorder with transducers zeroed toatmospheric pressure at the mid point of the thorax (e.g., see FIG. 3).Cardiac output (CO) was measured by thermal dilution as the average oftwo determinations injecting 5 ml of 0° C. Ringers lactate. Pulmonaryvascular resistance (PVR) and systemic vascular resistance (SVR) werecomputed by standard formulae; PVR measured at each inhaled NOconcentration is shown in FIG. 4. Appropriate statistical analyses wereperformed, and all data were expressed as mean ± standard error.

Administration of NO to lambs with pulmonary vasoconstriction induced byhypoxia

Five awake lambs were studied during a period of breathing a hypoxic gasmixture to induce acute hypoxic pulmonary hypertension. Three lambs wereexcluded due to sepsis and heart failure. Hemodynamic monitoringtechniques similar to those described above were used. We employed anon-rebreathing circuit containing a 25 liter reservoir bag and theF_(i) O₂ was reduced to 0.06-0.08 to produce a mean PAP near 25 mm Hg ata P_(a) O₂ near 30 mm Hg. Either 40 or 80 ppm NO was then added to theinspired gas mixture. Total gas flows were maintained at 35 1/min toprevent rebreathing due to hyperventilation. The inspired F_(i) O₂ wasmonitored with an electrode (model 5590, Hudson Co., Temecala, Calif.)and pure CO₂ was added to the inspired gas to maintain the end tidal CO₂concentration at 4.5-6%. Measurements of central hemodynamics and gasexchange were obtained at baseline, during hypoxia, and at 3 and 6minutes of NO breathing during hypoxia. Comparisons were performed usingpaired t-tests.

ii. Results

Two control lambs with no drug infusion breathed 80 ppm NO at an F_(i)O₂ of 0.6-0.7. There was no change of mean PAP, SAP, CO or SVR in theselambs.

In eight lambs regression analyses of NO concentration during U46619infusion vs. SVR, CO or mean SAP showed no significant change. However,all dose levels of NO inhalation produced a prompt reduction of thepulmonary vasoconstriction and pulmonary hypertension caused by U46619infusion (FIGS. 1, 2). The onset of pulmonary vasodilation occurredwithin seconds after beginning NO inhalation. The vasodilator effect wasnearly maximal within three minutes (FIG. 3). Ceasing to inhale NOcaused a return to the prior level of vasoconstriction within three tosix minutes. The inhaled NO pulmonary vasodilator response curve ofeight lambs is shown in FIG. 1. 5 ppm NO (an inhaled lung dose of 0.89μg/kg/min) significantly reduced the PA pressure, and an almost completevasodilator response occurred by inhaling 40 or 80 ppm. Afterconsidering the minor reduction over time of baseline PAP during U46619infusion, comparison of the vasodilator response of the second exposureto breathing 5, 10 and 20 ppm NO demonstrated no significant reductionfrom the prior series of exposures (FIG. 2). An additional study of fourlambs inhaling 80 ppm NO for one hour during U46619 infusiondemonstrated pulmonary vasodilation to a normal PAP, with pulmonaryhypertension recurring after NO inhalation.

All five lambs in which acute hypoxic pulmonary hypertension had beeninduced demonstrated a marked increase of cardiac output. In eachinstance when 40 or 80 ppm of NO was added to the inspired hypoxic gasmixture, pulmonary artery pressure returned to control levels despitethe maintenance of elevated cardiac output; mean PVR dropped 33% (Table1). The P_(a) O₂ and P_(v) O₂ during hypoxia with and without NO weresimilar.

                  TABLE 1                                                         ______________________________________                                        ALTERATIONS OF HEMODYNAMICS AND GAB EXCHANGE                                                                HYPOXIA +                                                                     40-80 PPM                                                 CONTROL  HYPOXIA    NO                                              ______________________________________                                        F.sub.i O.sub.2                                                                           0.21       0.06-0.08  0.06-0.08                                   P.sub.a O.sub.2 (mm Hg)                                                                   70.8 ±                                                                             4.4    28.2 ±                                                                           1.4* 31.1 ±                                                                           1.7*                              P.sub.v O.sub.2 (mm Hg)                                                                   36.8 ±                                                                             16.6   16.6 ±                                                                           1.8* 19.8 ±                                                                           3.2                               P.sub.a CO.sub.2 (mm Hg)                                                                  33.9 ±                                                                             1.4    38.6 ±                                                                           2.6  40.0 ±                                                                           2.7                               pHa         7.47 ±                                                                             0.01   7.42 ±                                                                           0.03 7.40 ±                                                                           0.03                              PAP (mm Hg) 16.7 ±                                                                             0.6    28.3 ±                                                                           2.2* 18.7 ±                                                                           1.1#                              LAP (mm Hg) 5.2 ±                                                                              0.8    6.4 ±                                                                            0.5  4.2 ±                                                                            1.0                               CO (l/min)  4.55 ±                                                                             0.13   7.08 ±                                                                           0.22*                                                                              7.56 ±                                                                           0.79*                             PVR (mm Hg/l/min)                                                                         2.51 ±                                                                             0.11   3.07 ±                                                                           0.25 2.01 ±                                                                           0.35#                             SAP (mm Hg) 103 ±                                                                              6      113 ±                                                                            7    106 ±                                                                            5#                                CVP (mm Hg) 3.0 ±                                                                              1.3    3.5 ±                                                                            0.8  2.8 ±                                                                            1.6                               SVR (mm Hg/l/min)                                                                         21.7 ±                                                                             1.4    16.2 ±                                                                           0.9* 13.7 ±                                                                           1.0*                              ______________________________________                                         n = 5, mean ± S.E.                                                         *p < .01 value differs from control                                           #p < .01 NO + hypoxia value differs from hypoxia                         

iii. Further Experiments

FIGS. 5a and 5b illustrate the ability of 180 ppm inhaled No to preventthe elevated PAP and PVR caused by the heparin-protamine reaction innine awake sheep as compared to control air-breathing sheep. Theheparin-protamine reaction was induced in these nine sheep by firstadministering heparin (200 U/kg; Elkins-Sinn, Cherry Hill, N.J.)followed five minutes later (at time zero) by protamine (2 mg/kg;Elkins-Sinn). Each of these sheep also served as a control. Sixadditional sheep were given an intravenous infusion of sodiumnitroprusside (40 μg/kg/min body weight; Elkins-Sinn) while breathingair (data not shown). The 180 ppm NO inhaled dose proved capable oflowering the heparin-protamine-induced PAP in this sheep model to adegree comparable to 40 μg/kg/min SNP infusion, and without the latterdrug's propensity to cause marked systemic hypotension.

Lungs from three lambs which had breathed 80 ppm NO for 180 min werestudied by light microscopy for evidence of morphological changes causedby breathing No. No significant differences between these lungs andcontrol lungs were observed.

B. Protocol for administration of gaseous NO to infants with PersistentPulmonary Hypertension of the Newborn

The following is a description of an approved experimental protocol forthe administration of NO to newborns at Massachusetts General Hospital.Selection of participants:

Ten patients with persistent pulmonary hypertension of the newborn(PPHN) will be enrolled in the study.

a. Inclusion criteria

infants under 1 week of age

infants with arterial blood sampling sites in the pre- and post-ductaldistribution

infants requiring mechanical ventilatory support

respiratory failure as defined by criteria of Short, Clin. Perinatol.14:737-748, 1987

infants may be receiving infusions of systemic vasodilators and/orbuffers (bicarbonate)

b. Exclusion criteria

prematurity as defined by a gestational age <37 weeks by examination,maternal-fetal ultrasound and dates

birth weight <2500 g

pulmonary hypoplasia as suggested by a history of oligohydramnios,congenital diaphragmatic hernia, congenital scoliosis, or featuresconsistent with asphyxiating thoracic dystrophy

unevacuated pneumothorax despite chest tube

pneumopericardium or pneumomediastinum with hypotension

fixed anatomic cardiac and vascular lesions (excluding isolated patentductus arteriosus and patent foramen ovale)

active pulmonary hemorrhage or platelet count <50, 000/mm³

cranial ultrasound within 24 hours of study entry providing evidence ofintracranial hemorrhage

hyperviscosity as defined by a venous hematocrit ≧70% within 24 hours ofbirth

sepsis, as defined by positive blood cultures for pathogenic organisms

those who do not have informed consent from a parent or legal guardian

Study procedure

Selected patients will be maintained in a supine position and willreceive 3 μg/kg fentanyl for sedation, and 0.1 mg/kg pancuronium bromidefor muscle relaxation (unless so treated within the previous hour). Theinfant will be transported to the catheterization suite accompanied byan attending pediatric anesthesiologist, where a flow directed pulmonaryartery catheter will be placed percutaneously via a femoral vein underlocal anesthesia. The catheter will directly measure pulmonary arterypressure in order to accurately assess the degree of pulmonaryhypertension and vasodilatory response to NO inhalation. Upon return tothe Neonatal ICU, the F_(i) O₂ will be adjusted to 0.90. The patientwill be allowed to equilibrate during this control phase for 20 minutesafter all necessary nursing and medical interventions have ceased. Ifimprovement, as defined below, has not occurred, an arterial bloodsample will be obtained from a post-ductal site. NO in nitrogen willthen be introduced into the breathing circuit by continuous flow. A oneway valve will prevent back flow of oxygen into the NO tank. The sameF_(i) O₂ (0.90) and flow rate will be maintained. The initialconcentration of inspired NO will be 20 ppm. Improvement will be definedas a P_(a) O₂ >100 mm Hg and a A-aDO₂ of <570 mm Hg (post-ductalsample). If no change is noted the concentration of inhaled NO will beincreased to 40 ppm at a constant F_(i) O₂ and flow rate. A post-ductalarterial blood gas will again be measured. If the same criteria areagain not met, the NO concentration will be increased to 80 ppm and athird arterial blood gas sampled. The breathing period for eachconcentration of NO will last 10 minutes.

Following termination of the treatment period, blood will again beobtained for arterial blood gas analysis. Samples will also be takenbefore and after No exposure for analysis of methemoglobin andhemoglobin levels and reticulocyte count. A blood smear will be examinedfor evidence of Heinz bodies. These will be repeated 24 hours aftertreatment to assess any changes associated with NO breathing. The totalvolume of blood sampled will be less than 5 ml.

Statistical methodology

Data will be assessed with an analysis of variance with repeatedmeasures of unequal group sizes. Winer, "Single factor experimentshaving repeated measures on the same elements", in StatisticalPrinciples in Experimental Design, 2d Ed., N.Y., McGraw-Hill, (1971),pp. 261-308. Post hoc testing will be with a Mann-Whitney U.Significance will be judged at the 5% level.

C. Results of administering NO to infants with persistent pulmonaryhypertension of the newborn (PPHN)

First subject

Through compassionate use, nitric oxide was administered to an infantsuffering from persistent pulmonary hypertension and congenital heartdisease. As a result of prolonged ventilation, absence of a preductalarterial blood sampling site, and the existence of theatrial-ventricular (AV) canal, the patient was not included in the PPHNstudy mentioned above.

The patient was a 3225 gm, full term male who had been treated withextracorporeal membrane oxygenation (ECMO) because of the severity ofhis congenital heart disease and profound hypoxemia. He had been takenoff ECMO and was being maintained intubated and ventilated in thenewborn intensive care unit. He subsequently became progressivelyhypoxemic, as reflected in his post-ductal pulse oximetry (POX) values.By the time he was taken to the catheterization laboratory to confirmthe existence of the A-V canal and to determine if some emergent cardiacsurgery was needed, he was receiving maximal medical and ventilatorylife support and remained dangerously hypoxemic. Under thesecircumstances, we were granted consent to treat the patient with nitricoxide.

Upon arrival to the catheterization laboratory, the patient wasextremely cyanotic. He was treated with fentanyl, oxygen,hyperventilation and intravenous fluid boluses to stabilize him prior toadministering NO. As shown in Table 2, the catheterization revealedsevere pulmonary hypertension and an A-V canal. The shunting did notappear to correct with treatment with oxygen or hyperventilation.

                                      TABLE 2                                     __________________________________________________________________________    HEMODYNAMICS AND BLOOD GAS VALUES FOR                                         NO INHALATION TREATMENT OF INFANT WITH PPHN                                                  F.sub.I O.sub.2                                                                  F.sub.I O.sub.2                                                                  NO  NO  NO  OFF                                                                              NO  OFF                                            ARRIVAL                                                                             1.0                                                                              0.9                                                                              20 ppm                                                                            40 ppm                                                                            80 ppm                                                                            #1 80 ppm                                                                            #2                                    __________________________________________________________________________    O.sub.2 SAT (%)                                                               RA       23    61 67 67  72  74  14 --  --                                    PA       28    69 72 70  74  75  17 --  --                                    POSTDUCTAL                                                                    ART      63    74 84 85  74  88  28 85  19                                    POX      --    89 91 91  93  94  21 90  24                                    POSTDUCTAL                                                                             30    43 48 46  50  51  21 48  16                                    ARTERIAL                                                                      PO.sub.2 (mmHg):                                                              ART                                                                           MEAN                                                                          PRESSURE                                                                      (mmHg)                                                                        RA        6    4  4  5   4   5   -- --  --                                    PA       57    52 47 50  52  53  -- --  --                                    ART      52    50 45 45  43  47  -- --  --                                    __________________________________________________________________________     POX = pulse oximeter                                                     

We utilized a regulator to step-down the pressure of the NO into ablender, which allowed us to adjust the relative amounts of the 800 ppmNO/N₂ and 100% N₂ supplies. Treating the patient with pure oxygen, weincreased the flow of N₂ through a flow regulator into the inspiratorycircuit of the breathing circuit of the breathing circuit until theF_(I) O₂ was 0.9. The effects are shown in Table 2. This provided a 1:10dilution of the nitrogen gas. We then used the blender to adjust therelative amounts of N₂ and NO/NO₂ to provide 0 to 80 ppm of NO.

The data in Table 2 demonstrate that exposure to NO had no adverseeffect on systemic blood pressure ("Mean Pressure-Art"), while inducinga modest increase in arterial saturation, pulse oximetry values, andarterial partial pressure of oxygen. This may reflect a stabilizingeffect of the gas during this period. After the nitric oxide wasdiscontinued and the central catheters were removed, the arterialsaturation and oxygen gas tension precipitously dropped. The RA and PAvalues could not be determined, as the catheters had been removed. Asother attempts to resuscitate the patient were failing, the nitric oxidewas restarted in an attempt to improve the baby's condition. Itsucceeded in improving the oxygen saturation and blood gas tension. In asubsequent attempt to wean the patient off nitric oxide, again thepatient's oxygenation level deteriorated to dangerously low levels. Thepatient was maintained on nitric oxide and returned to the newbornintensive care unit.

While in the intensive care unit, prostaglandin E1 was infused into thepatient in an attempt to dilate the pulmonary vasculature. Despite astandard dosage of prostaglandin, nitric oxide could not be discontinuedwithout the return of dangerously low oxygen saturations. The patientremained on nitric oxide until he could be placed on ECMO. This trialdemonstrated the utility of nitric oxide in improving gas exchange inthis patient with pulmonary hypertension and congenital heart disease.

Subsequent subjects

Two more infants with PPHN have been treated by NO inhalation. Both hadan excellent response to breathing NO at 20-80 ppm, showing increases inpreductal oxygenation, and both survived longterm. One of the infantsshowed such rapid improvement with NO inhalation alone that ECMO wasaltogether avoided.

D. Results of administering NO to adults with Adult Respiratory DistressSyndrome

First subject

The patient, a 42-year old woman, had suffered for three weeks fromadult respiratory distress syndrome (ARDS) due to aspiration pneumonia.There was diffuse pulmonary edema and a large Q_(VA) /Q_(T) (30%). After21 days of venovenous extracorporeal membrane oxygenator support (3liters/min blood flow), the mean PAP was 55 mm Hg.

The short term effects of inhaled nitric oxide were compared with thoseof i.v. prostacyclin (PGI₂ ; 5 ng/kg/min). Mean pulmonary arterialpressure (PAP), right ventricular ejection fraction (RVEF) and gasexchange variables were evaluated. RVEF was assessed by thermodilution,and gas exchange alterations were analyzed using the multiple inert gaselimination technique (MIGET). MIGET and RVEF data were obtained on twodifferent occasions. Ventilator settings were tidal volume 6 ml/kg,respiratory rate 14/min, F_(i) O₂ 0.4-0.48 and 5 cm H₂ O of PEEP(positive end expiratory pressure).

                  TABLE 3                                                         ______________________________________                                        HEMODYNAMIC RESULTS OF TREATMENT OF ADULT                                     WITH PULMONARY HYPERTENSION                                                                  Con-    NO      NO                                                       PG12 trol    18 ppm  36 ppm                                                                              Control                                  ______________________________________                                        #1  PAP(mm Hg)  46     54    42    37    49                                       PCWP(mm Hg) 12     15    15    15    14                                       MAP(mm Hg)  81     86    78    75    80                                       PaO.sub.2 (torr)                                                                          74     104   146   127   100                                      Q.sub.A /Q.sub.T %                                                                        57     38    26    33    30                                       low V.sub.D /Q %                                                                           0      2     1     0     0                                       V.sub.D /V.sub.T %                                                                        51     47    43    40    41                                   #2  PAP(mm Hg)  42     52    38    36    50                                       PCWP(mm Hg) 14     14    14    12    14                                       MAP(mm Hg)  86     91    88    86    88                                       PaO.sub.2 (torr)                                                                          81     84    127   113   90                                       RVEF %      42     27    36    39    28                                   ______________________________________                                    

As illustrated in FIG. 6 and in Table 3, inhaled NO lowered PAP andimproved RVEF as did i.v. PGI₂, but, in contrast to PGI₂, NO increasedPaO₂ and decreased right-to-left shunt and V_(D) /V_(T). Inhalation of18 ppm NO in oxygen caused a reduction of mean PAP to 38-42 mm Hg (adecrease of 12-14 mm Hg) and reduced the PVR by 44%, the wedge pressureremaining constant near 15 mm Hg and the cardiac output near 7liters/min and unchanged. There was a small additional vasodilation (2-5mm Hg) caused by increasing the NO concentration to 36 ppm. Vasodilationwith NO was sustained for about 11/2 hours, when administration waselectively ceased. During NO inhalation, the Q_(VA) /Q_(T), measuredwith sulphur hexafluoride, decreased from 38% to 26% (18 ppm NO) and 33%(36 ppm NO). There was no change of systemic arterial pressure withinhaled NO: unlike the systemic vasodilator PGI₂, which increased Q_(VA)/Q_(T) to 57%, inhaled NO predominantly vasodilates the vasculature ofventilated lung regions. This trial is a clear demonstration of theselective ability of low levels (18-36 ppm) of inhaled NO to act as apotent pulmonary vasodilator in a patient with severe acute lung injury(ARDS), without increasing the shunt.

Subsequent subjects

Nine additional patients have been treated for ARDS by NO inhalation,for periods up to 28 days. Seven survived in spite of their severerespiratory distress symptoms, displaying marked reductions of Q_(VA)/Q_(T) during NO breathing, as well as a reduced PAP. No importantincrease of methemoglobin levels was observed. These results indicatedthat NO inhalation for up to several weeks is a promising therapy oracute respiratory failure.

E. Results of administering NO to humans with normal (non-constricted)and hypoxic (constricted) lungs

The effects of breathing 40 ppm NO were studied in five awake, healthyhuman volunteer subjects inhaling various gas mixtures for 10 minperiods, with measurements starting at 6 min. Table 4 shows that insubjects breathing air with a normal (21% v/v) O₂ concentration, andwhose lungs therefore were not vasoconstricted, NO has no pulmonary orsystemic vasodilatory effect.

                  TABLE 4                                                         ______________________________________                                        EFFECTS OF 40 PPM NO ON THE NON-CONSTRICTED                                   HUMAN LUNG                                                                              Air      Air (21% O.sub.2) +                                                                      Air                                                       (21% O.sub.2)                                                                          40 ppm NO  (21% O.sub.2)                                   ______________________________________                                        PAP mmHg    13.7 ±                                                                             1.7    14.0 ±                                                                           1.8  15.4 ±                                                                           2.8                               PCWP mmHg   9.1 ±                                                                              1.7    10.1 ±                                                                           2.5  9.9 ±                                                                            2.2                               CO l/min    6.40 ±                                                                             0.92   6.40 ±                                                                           0.88 6.95 ±                                                                           1.18                              PVR mmHg · min/l                                                                 0.72       0.61       0.79                                        MAP mmHg    87.4 ±                                                                             6.0    88.0 ±                                                                           3.7  90.2 ±                                                                           5.4                               CVP mmHG    5.7 ±                                                                              1.4    6.3 ±                                                                            1.7  6.1 ±                                                                            1.6                               PaO.sub.2 mmHg                                                                            99.6 ±                                                                             7.5    94.7 ±                                                                           16.3 95.3 ±                                                                           14.5                              PaCO.sub.2 mmHg                                                                           38 ± 6      38 ±                                                                             5    39 ±                                                                             4                                 SaO.sub.2 % 97.6 ±                                                                             0.4    96.0 ±                                                                           1.0  97.1 ±                                                                           1.2                               ______________________________________                                         Values given as X ± S.D. n = 5                                        

In contrast, the same subjects breathing a relatively low level ofoxygen (12% v/v) exhibited hypoxia-induced pulmonary vasoconstrictionwith elevated PAP and PVR, an effect that could be reversed completelyby adding 40 ppm NO to the inhaled gas mixture (Table 5).

                                      TABLE 5                                     __________________________________________________________________________    EFFECTS OF 40 PPM NO ON THE HYPOXIC,                                          VASCONSTRICTED HUMAN LUNG                                                                  Air         12% O.sub.2 Air                                                   (21% O.sub.2)                                                                       12% O.sub.2                                                                         40 ppm NO                                                                           12% NO                                                                              (21% NO)                                 __________________________________________________________________________    PAP  mmHg    14.3 ± 2.3                                                                       19.1 ± 2.6#                                                                      13.7 ± 1.7*                                                                      15.7 ± 2.2                                                                       14.5 ± 1.5                            PCWP mmHg    8.8 ± 1.9                                                                        8.5 ± 1.3                                                                        8.5 ± 2.2                                                                        9.2 ± 1.6                                                                        9.7 ± 1.9                             CO   l/min   6.65 ± 0.95                                                                      8.66 ± 1.87                                                                      8.37 ± 1.68                                                                      8.5 ± 1.9                                                                        7.06 ± 1.84                           PVR  mmHg · min/l                                                                 0.83  1.22  0.62  0.76  0.68                                     MAP  mmHg    88.8 ± 6.9                                                                       89.4 ± 8.4                                                                       86.0 ± 5.7                                                                       84.4 ± 7.6                                                                       88.4 ± 6.3                            CVP  mmHg    5.9 ± 3.0                                                                        5.6 ± 2.2                                                                        5.2 ± 2.6                                                                        5.0 ± 1.9                                                                        6.2 ± 1.6                             PaO.sub.2                                                                          mmHg    99 ± 14                                                                          47 ± 5                                                                           45 ± 5                                                                           45 ± 8                                                                           93 ± 16                               PaCO.sub.2                                                                         mmHg    40 ± 4                                                                           35 ± 3                                                                           34 ± 5                                                                           33 ± 6                                                                           39 ± 6                                SaO.sub.2                                                                          %       97.5 ± 1.0                                                                       85.4 ± 3.4                                                                       83.9 ± 5.7                                                                       82.6 ± 11                                                                        96.8 ± 1.3                            __________________________________________________________________________     n = 5,                                                                        X ± S.D.                                                                   #p < 0.01 value differs from value in first column                            *p < 0.01 value differs from the previous situation                      

2. AIRWAY SMOOTH MUSCLE DILATION

A. Methods

Animal preparation

Male Hartley strain guinea pigs (300-440 g body wt) were anesthetizedwith α-chloralose (50 mg/kg) and urethane (500 mg/kg) (Drazen et al., J.Appl. Physiol. 48:613-618, 1980). A tracheostomy was performed, and theanimals were intubated with a tubing adaptor (ID 1.65 mm) and ventilatedwith a small animal ventilator (Harvard Apparatus, a division of EalingScientific, Natick, Mass.) at 8 ml/kg and 60 breaths/min. A jugular veinwas cannulated for intravenous administration of drugs. The chest wasopened by bilateral excision of a portion of the ribs anteriorly so thatthe lungs were exposed to atmospheric pressure (Shore and Drazen, J.Appl. Physiol. 67:2504-2511, 1989). A positive end expiratory pressureof 3-4 cmH₂ O was provided.

Material

Guinea pigs were then placed inside a plethysmograph (Amdur and Mead,Am. J. Physiol. 192:363-368, 1958), that was connected to a largereservoir containing copper mesh to maintain the plethysmographisothermal. Plethysmograph pressure was measured with a differentialpressure transducer (Celesco, Canoga Park, Calif.); the opposite side ofthis transducer was connected to a similar reservoir. Pressure at theairway opening was measured from a side tap in the tracheal canula.Transpulmonary pressure was measured with a differential pressuretransducer (Celesco) as the difference between airway opening pressureand the pressure inside the plethysmograph. Flow was obtained byelectrical differentiation of the volume (plethysmograph pressure)signal. Tidal volume was measured by recording the pressure changes inthe body plethysmograph. Volume, flow, and transpulmonary pressuresignals were recorded on a strip chart (General Scanning, Watertown,Mass.). Pulmonary resistance and dynamic compliance were calculated by acomputer program according to the method of von Neergard and Wirz (Z.Klin. Med. 105:35-50, 1927; Z. Klin. Med. 105:52-82, 1927).

The apparatus and conditions used are diagrammed in FIG. 7. The inspiredgas was a precise mixture of nitrogen and oxygen blended via a Y piecetube and immediately diluted with nitric oxide (NO) to produce thecorrect inspired concentration in a 5 liter gas mixture bag. Withvolumetrically calibrated flowmeters, varying quantities of NO mixedwith N₂ were substituted for pure N₂ to obtain the desired NOconcentration at an inspired oxygen concentration (FIO₂) of 0.30-0.32.The total inflow gas rate was maintained at 2.5 l/min. The gas mixturewas then sent via a 3 cm ID tube filled with 90 ml of soda lime toscavenge nitrogen dioxide (Stavert and Lehnert, Inhal. Toxicol. 2:53-67,1990), then through a filter before the ventilator. Just after theventilator inflow tube, a vacuum was adjusted to maintain the gasmixture bag nearly empty and continuously drive fresh gas into theventilator circuit. The expiratory gas from the ventilator was scavengedwith a vacuum and set up to maintain a positive end expiratory pressureof 3-4 cm H₂ O. NO was obtained from Air Products and Chemicals, Inc.(Allentown, Pa.) as a mixture of 1,034 ppm NO in pure nitrogen. Achemiluminescence NO/NO_(x) analysis (Fontijin et al., Anal. Chem.42:575-579, 1970) was performed before and after the soda lime filledtube, and just before the inspiratory valve of the ventilator (see FIG.7) to assess the nitrogen dioxide concentration and adjust theflowmeters to provide the different levels of NO concentration.

Protocol

Twenty-four guinea pigs were studied. Three series of studies werecompleted on three separate groups of animals.

Group A

Nine guinea pigs were included in 3 sets of measurements.

i. NO effects on normal bronchial tone

After baseline measurements of tidal volume, lung resistance and dynamiccompliance, the effects on baseline bronchial tone of inhaling 300 ppmNO at FIO₂ 0.30-0.32 for 6 to 10 minutes were evaluated (FIG. 8).

ii. Dose-response study of intermittent NO inhalation duringmethacholine infusion

After baseline measurements, the same guinea pigs were given anintravenous infusion of a potent bronchoconstrictor, methacholine, at arate of 2.5-7.5 μg/kg/min in order to reach a medium level ofbronchoconstriction (3 to 4 fold the baseline lung resistance). After astable period, each animal was ventilated with a series of gas mixturesof 5, 10, 25, 50, 100 and 300 ppm NO for 10 minutes at constant FIO₂(0.30-0.32). After each level of NO exposure, lungs were inflated tototal capacity to minimize the effects of airway closure. A secondexposure to 10 and 50 ppm NO for 10 minutes was performed, and eachguinea pig was examined for the occurrence of acute tolerance. After thelast level of NO ventilation, methacholine infusion was stopped andmeasurements done after a stable period of lung mechanics to obtain thereference point for the dose-response study. Only then were the lungsinflated to total lung capacity to reach a stable new baseline value(see FIGS. 9-12).

iii. Study of tolerance to 1 hour of NO inhalation during methacholineinfusion

Guinea pigs were given an infusion of methacholine to raise bronchialtone 3 to 4 fold, after which the animals were ventilated with a 100 ppmNO gas mixture for 1 hour at FIO₂ 0.30-0.32. Repeated airwaymeasurements were obtained every 5 minutes and then 5 and 10 minutesafter ceasing NO inhalation. Methacholine infusion was then discontinuedand repeated measurements were obtained after a stable period of lungventilation, and once again after lung inflation to total lung capacity.Methemoglobin levels were measured (Zwart et al., Clin Chem27:1903-1907, 1981) at the time of the surgical procedure and againafter the tolerance study (FIG. 13).

Group B

Ten guinea pigs were included in 2 sets of experiments.

i. Study of tolerance of 80 minutes of methacholine infusion alone

To evaluate the stability of this bronchoconstrictor model, guinea pigswere given an infusion of methacholine at a rate of 2.5-7.5 μg/kg/min toreach the same level of bronchoconstriction as in the 1 hour NOinhalation study (see FIG. 13). Animals were ventilated with anoxygen/nitrogen gas mixture at constant FIO₂ (0.30-0.32). Repeatedmeasurements were obtained every 5 minutes. At 10 and 70 minutes,flowmeters were adjusted to simulate NO ventilation. Methacholineinfusion was then discontinued. Repeated measurements were obtainedafter a stable period of lung mechanics, and once again after lunginflation to total lung capacity.

ii. Study of co-regulation of airway smooth muscle tone by cyclic-AMP-and cyclic-GMP-dependent mechanisms

After baseline measurements, 5 guinea pigs were given a methacholineinfusion to raise their lung resistance to the medium level ofbronchoconstriction. The guinea pigs received first a terbutalineaerosol followed 10 minutes later by a 100 ppm NO inhalation for 6minutes, while maintaining a constant FIO₂ (0.30-0.32). The terbutalineaerosol was given as follows: 4 ml of a 40 μg/ml terbutaline solutionwas placed in the reservoir of a nebulizer (Respigard II) and driven by4 l/min air. The nebulizer was connected via a stopcock to the Y pieceof the ventilator circuit and to a tube immersed in 3-4 cm water. At thetime of the nebulization, the ventilator was disconnected so that thenebulizer circuit was connected to the airway and 20 nebulized breathsof terbutaline at the same tidal volume were given. Then the ventilatorwas reconnected, and the nebulizer disconnected. At the end of thestudy, methacholine infusion was discontinued until stable lungmechanics had returned, and then the lungs were inflated to total lungcapacity to reach a final baseline value. Repeated respiratory mechanicsmeasurements were obtained and every 2 minutes during the NO andterbutaline periods (FIGS. 14 and 15).

Group C

Study of S-nitroso-N-acetylpenicillamine (SNAP) during methacholinebronchoconstriction

SNAP was prepared according to the method described in Field et al., J.Chem. Soc. Chem. Comm. (1978), 249-250, and was stored as crystals at 0°C. for up to 120 days without detectable degradation (as assayed byabsorbance at 595 nm).

After obtaining baseline respiratory measurements, 5 guinea pigs weregiven a methacholine infusion to raise their lung resistance to a mediumlevel of bronchoconstriction. After two minutes, each guinea pigreceived a SNAP aerosol. The SNAP aerosol was given as follows: 200 mMof SNAP dissolved in an ethanol/water mixture (4 ml) was placed in thereservoir of a nebulizer (Respigard II) and driven by 4 l/min air. Thenebulizer was connected via a stopcock to the Y piece of the ventilatorcircuit and to a tube immersed in 4 cm water. At the time ofnebulization, the ventilator was disconnected so the nebulizer circuitwas connected to the airway and 20 nebulized breaths of SNAP at the sametidal volume were given. Then the ventilator was reconnected and thenebulizer disconnected. At the end of the study (15 minutes) themethacholine infusion was discontinued until stable lung mechanics hadreturned; then the lungs were inflated to total lung capacity to reach afinal baseline value. Repeated respiratory mechanics measurements wereobtained every two minutes (FIG. 16).

B. Results

Inhalation of nitric oxide-containing gas mixtures produced aconsistent, rapid and profound reduction of lung resistance and anincrease of lung compliance (FIGS. 9-12). Onset of dilation was rapid,beginning within a few seconds after inhalation. Nitric oxide inhalationreversed the profound bronchoconstriction caused by methacholineinfusion, but also decreased the baseline bronchomotor tone of theanesthetized guinea pig without a methacholine infusion (FIG. 8). Nitricoxide inhalation produced bronchodilation at very low doses (5 ppm),although a greater and more rapid reduction of airway resistance wasobtained at 100 or 300 ppm NO (FIGS. 10, 11 and 12). Complete reversalof methacholine bronchoconstriction occurred at 300 ppm NO. There was notolerance produced by NO breathing, since breathing 100 ppm NOeffectively and stably reduced the airway resistance for one hour (FIG.13). Methemoglobin levels remained below 5% after one hour of breathing100 ppm NO. This model of producing airway constriction by methacholineinfusion produced stably increasing levels of airway resistance for upto one hour (see FIG. 13), establishing the reliability andreproduceability of the above-described studies on the efficacity of NOas a bronchodilator.

During a methacholine infusion, the bronchodilating effects of No areadditive with the effects of inhaling a commonly nebulizedbronchodilator, the β₂ agonist, terbutaline (FIG. 14). We have observedthis additive bronchodilating effect to occur whether NO gas isadministered before (FIG. 14) or after (FIG. 15) terbutaline. SNAP, anitric oxide donor molecule, was nebulized for 20 breaths into theairways of 5 methacholine-bronchoconstricted guinea pigs. In each animala prompt and profound reduction of lung resistance was produced whichlasted about 15 minutes (FIG. 16). Thus, inhalation of NO donorcompounds can also produce bronchodilation.

3. PROLONGATION OF ACTION OF INHALED NO BY PDE INHIBITOR

Both nitric oxide (NO) and endothelium-derived relaxing factor (EDRF)are produced from L-arginine by nitric oxide synthases (NOS). It hasbeen proposed that, once liberated from endothelial cells, NO activatessoluble guanylate cyclase and produces vasorelaxation by inducing anincrease of guanosine-3',5'-cyclic monophosphate (cGMP) levels insubadjacent smooth muscle cells (Ignarro, Ann. Rev. Pharmacol. Toxicol30:535-60, 1990; Ignarro, Circ. Res. 65:1-21, 1989; Johns, J.Cardiothorac. Vasc. Anesth. 5:69-79, 1991; Johns, (editorial)Anesthesiology 75:927-931, 1991; Ignarro, Biochem. Pharmacol.41:485-490, 1991; Moncada et al., Pharmacol. Reviews 43:109-142, 1991).Zaprinast™ (M&B 22948; 2-o-propoxyphenyl-8-azapurin-6-one, Rhone-PoulencRorer, Dagenham Essex, UK) selectively inhibits the hydrolysis of cGMPwith minimal effects on the breakdown of adenosine 3',5'-cyclicmonophosphate (cAMP) in vascular smooth muscle cells in isolatedvascular rings (Trapani et al, J. Pharmacol. Exp. Ther. 258:269-274,1991; Harris et al., J. Pharmacol. Exp. Ther. 249:394-400, 1989; Lugnieret al., Biochem. Pharmacol. 35(10):1743-1751, 1986; Souness et al., Br.J. Pharmacol. 98:725-734, 1989). It was therefore tested as a model PDEinhibitor for use in prolonging the pharmaceutical effects of inhaled NOin animals.

Materials and Methods

These investigations were approved by the Subcommittee for ResearchAnimal Care of the Massachusetts General Hospital, Boston.

Animal Preparation

Nine Suffolk lambs weighing 20-25 kg were anesthetized by inhalation ofhalothane in oxygen. Their tracheas were intubated and their lungsmechanically ventilated at 15 breaths/minute and 15 ml/kg tidal volumewith a large animal ventilator (Harvard Apparatus, Natick, Mass.). A 7Fthermodilution pulmonary artery catheter (Edwards Lab, Santa Anna,Calif.) was placed via the right external jugular vein through an 8Fintroducer (Cordis, Miami, Fla.). The femoral artery was cannulated witha polyvinyl chloride catheter (2 mm ID) advanced 30 cm into the aortafor continuous arterial pressure monitoring and arterial blood sampling.A tracheostomy was performed and an 8.0 mm ID cuffed tracheostomy tube(Portex, Keene, N.H.) was inserted to allow for spontaneous ventilation.Studies began three hours after emergence from the anesthesia when thefollowing exclusion criteria did not occur: a peripheral white bloodcell count less than 4,000 or more than 12,000/mm³, mean PAP more than20 mmHG, or a core temperature of more than 40.1° C. The lambs werehoused in a Babraham cage with access to food and water.

Hemodynamic Measurements

Systemic arterial pressure (SAP), pulmonary arterial pressure (PAP), andcentral venous pressure (CVP) were measured continuously and pulmonaryartery wedge pressure (PCWP) was measured intermittently usingcalibrated pressure transducers (Cobe Laboratories, Lakewood, Colo.)zeroed at the mid-chest level and continuously recorded on a thermalchart recorder (Western Graphtec, Inc., Marck 10-1, Irvine, Calif.).Thermodilution cardiac output (CO) was measured as the average of twodeterminations after injection of 5 ml 0° C. Ringer's lactate. Pulmonaryvascular resistance (PVR) and systemic vascular resistance (SVR) werecomputed by standard formulae. The change of mean PAP (ΔPAP) from thebaseline level of U46619-induced pulmonary hypertension was calculatedby subtracting the mean PAP during NO inhalation from the baseline levelpulmonary hypertension. The duration of the vasodilating response toinhaled NO was determined by measuring the elapsed time from thediscontinuation of NO inhalation until mean PAP returned to itspre-inhalation baseline value, and was expressed as the half time of theresponse (t1/2).

NO Delivery and Measurement

During the study, the tracheostomy was connected to a circuit consistingof a 5 liter reservoir bag and a two-way non-rebreathing valve (HansRudolph, Inc., Kansas City, Mo.) to separate inspired from expired gas.Expired gas was scavenged and discarded. Oxygen and nitrogen was mixedto produce FlO₂ of 0.6-0.7. Nitric oxide gas (800 ppm in N₂, Arico,Riverton, N.J.) was introduced into the inspiratory limb of thebreathing circuit immediately before the reservoir bag. The FlO₂ wasmeasured (oxygen meter No. 5590, Hudson, Temecula, Calif.) distal toreservoir bag after the NO-containing gases were mixed. Theconcentration of NO was continuously measured by chemiluminescence(model 14A, Thermo Environmental Instruments, Inc., Franklin, Mass.;Fontijin et al., Anal. Chem. 42:575-579, 1970) at the inspiratory sideof the one way valve. The exhaled gases, as well as those dischargedfrom the chemiluminescence analyzer, were scavenged by use of a Venturlexhalation trap maintained at negative atmospheric pressured by thelaboratory's central vacuum system. The ambient NO/NO₂ levels, asmeasured intermittently by chemiluminescence, did not increase duringthe experiments.

Measurements of Plasma cGMP Levels

Cyclic GMP (cGMP) levels were determined using ¹²⁵ I radioimmunoassay(Biomedical Technologies, Inc., Stoughton, Mass.) according to themethodology of Harper and Brooker (Harper et al., J. Cyclic NucleotideRes. 1:207-218, 1975). Briefly, 10 μl of 50 mM isobutylmethylxanthine(IBMX) was added to 1 ml of citrated blood and the mixture wascentrifuged at 2500×g and 4° C. for 10 minutes. The supernatant wasdiluted with acetate buffer and acetylated with acetic anhydrate andtriethylamine mixture. Subsequently, cGMP concentrations in the sampleswere determined based on the competitive binding of sample and knownamounts of ¹²⁵ I-cGMP for a specific antibody. All measurements wereduplicated and the intra- and inter-assay quality were controlled bymeasuring known amount of cGMP. The cGMP concentration in the bloodsamples were expressed as picomoles cGMP per milliliter plasma.

Protocol

A. Dose-response study of intermittent NO inhalation during U46619infusion without and with Zaprinast infusion. Six lambs were studiedwhile spontaneously breathing at FlO₂ 0.6-0.7. After baselinemeasurements were made, a potent pulmonary vasoconstrictor, the stableendoperoxide analogue of thromboxane (5Z=9α, 13E,15S)-11,9,-(Epoxymethano) prosta-5,13-dien-1-oic acid (U46619, Upjohnand Cayman chemical) was infused via the external jugular catheter. Theinfusion rate (0.5-1.0 μg-kg⁻¹ min⁻¹) was titrated to achieve a mean PAPof 30 mmHg. After 10 minutes of steady state pulmonary hypertension andhemodynamic measurements, each of the six lambs breathed in random ordera series of NO/oxygen mixtures of 5, 10 and 20 ppm NO for 6 minutes.Each NO exposure was followed by the period of breathing without NOuntil the mean PAP returned to previous baseline hypertensive value.Hemodynamic measurements were recorded at 3 and 6 minutes during NOinhalation and repeated every 3 minutes after discontinuing NOinhalation. Arterial blood samples were drawn every 6 minutes during thestudy to determine the plasma cGMP levels. The U46619 infusion was thenstopped and the lambs were allowed to recover. After a 30-minuterecovery period and repeat baseline measurements, a loading dose ofZaprinast (2 mg-kg⁻¹ over 5 minutes) was administered followed by aZaprinast infusion (0.1 mg-kg⁻¹ min-⁻¹). Twenty minutes later, pulmonaryhypertension was again induced by the intravenous infusion of U46619.Once steady state pulmonary hypertension was established, the rates ofinfusion of the both drugs were kept constant until the end of thestudy. The dose of U46619 (1.1-3.6 μg-kg⁻¹ min⁻¹) needed to achieve thesame degree of pulmonary hypertension during the Zaprinast infusion wasgreater than without Zaprinast. After 10 minutes of steady state andrepeat hemodynamic measurements, the lambs breathed NO as describedabove. The order of NO inhalation was same before and during theZaprinast infusion. The purpose of this randomization was to avoid theeffects of possible concentration changes of Zaprinast. Hemodynamicvariables were measured every 3 minutes throughout the study period.Plasma levels of cGMP were measured at 3 and 6 minutes during NOinhalation and every 6 minutes during the recovery period.

B. Transpulmonary difference of plasma cGMP concentration during NOinhalation without and with Zaprinast. Two additional lambs were studiedto determined the amount of cGMP produced in the lung and released intothe pulmonary venous blood during NO inhalation. The lambs were givenNO/oxygen mixtures in an increasing order (0.1, 1.0, 5.0, 10, 20 ppm)after stable pulmonary hypertension was established by U46619 infusion.This order was adopted to avoid accumulation of plasma cGMP becausesignificantly increased plasma concentrations of cGMP were found only on20 ppm of NO inhalation in the protocol A. The NO inhalation wasrepeated during the Zaprinast infusion in the same order. Pulmonaryarterial and aortic blood samples were drawn simultaneously during eachNO inhalation and 3 minutes before the next NO inhalation duringbaseline pulmonary hypertension.

C. A demonstration of intermittent NO inhalation. In one additionallamb, the effects of intermittent NO inhalation with and withoutZaprinast during U46619 induced pulmonary hypertension were studied.After stable baseline pulmonary hypertension was established by U46619infusion, the lamb inhaled 40 ppm NO for 4 minute periods. The U46619infusion was then discontinued. After a 30-minute recovery period,Zaprinast was administered as described above and pulmonary hypertensionwas re-established by U46619 infusion. Nitric oxide (40 ppm) was inhaledfor 4 minutes. Subsequently, the 4 minute exposure was repeated eachtime the ΔPAP decreased by 50 percent.

Chemicals

Zaprinast (2-o-propoxyphenyl-8-azapurin-6-one) was a generous gift fromRhone-Poulenc Roler (Dagenham, Essex, UK). The stock solution ofZaprinast was prepared in 0.05N NaOH. This stock was diluted withRinger's lactate to a final concentration of 8 mg-ml⁻¹ just before use.Immediately before the study, 5 mg of U46619 was dissolved in 50 ml ofRinger's lactate.

Data Analysis

The changes of mean PAP and PVR are expressed as the difference betweenthe stable baseline pulmonary hypertension value and the lowest valuerecorded during each NO inhalation. The half time of the vasodilatorresponse was determined by measuring the elapsed time from thetermination of each NO inhalation to when the mean PAP returned to avalue half-way between the lowest mean PAP value recorded during NOinhalation and the baseline pulmonary hypertension value. All the dataare presented as mean ±SE. The data were analyzed using a paired t-testor an analysis of variance (ANOVA) with repeated measures. P<0.05 wasused as the criterion for statistical significance.

Results

A. Dose-Response Study of Intermittent NO Inhalation During U46619Infusion Without and With Zaprinast

The mean PAP change (ΔPAP) during NO inhalation is shown in FIG. 19. Atall dose levels, there was no difference between NO inhalation with orwithout Zaprinast. The duration of the vasodilating response to inhalednitric oxide (t1/2) was increased by the Zaprinast infusion at all NOdoses (FIG. 1B). There was no significant difference in SVR or CObetween NO inhalation with or without Zaprinast (Table 6). Compared withits control, PVR decreased slightly with Zaprinast, but this change wasnot statistically significant (Table 6). This may be due to a slightincrease of CO by the Zaprinast infusion (Table 6), since mean PAP wasnot decreased by Zaprinast and PCWP was stable throughout (data notshown).

                                      TABLE 6                                     __________________________________________________________________________    SVR               PVR         CO                                              (mmHg · 1.sup.-1 · min.sup.-1)                                                (mmHg · 1.sup.-1 · min.sup.-1)                                          (l · min.sup.-1)                       n = 6 Control                                                                             Zaprinast                                                                           Control                                                                             Zaprinast                                                                           Control                                                                             Zaprinast                                 __________________________________________________________________________    Baseline                                                                            16.6 ± 2.01                                                                      15.3 ± 1.13                                                                      1.39 ± 0.26                                                                      1.30 ± 0.22                                                                      5.67 ± 0.92                                                                      5.98 ± 0.57                            PHTN  33.0 ± 3.01                                                                      41.6 ± 4.93                                                                      6.15 ± 0.86                                                                      6.23 ± 1.11                                                                      3.35 ± 0.42                                                                      2.72 ± 0.33                            (U46619)                                                                      NO 5 ppm                                                                            36.3 ± 4.55                                                                      33.0 ± 4.43                                                                      4.62 ± 0.71                                                                      2.99 ± 0.75                                                                      2.94 ± 0.28                                                                      3.67 ± 0.47                            NO 10 ppm                                                                           35.1 ± 4.81                                                                      34.9 ± 6.04                                                                      3.78 ± 0.92                                                                      2.49 ± 0.50                                                                      3.08 ± 0.29                                                                      3.55 ± 0.44                            NO 20 ppm                                                                           35.1 ± 3.40                                                                      33.7 ± 6.10                                                                      3.01 ± 0.48                                                                      1.95 ± 0.56                                                                      2.92 ± 0.19                                                                      3.73 ± 0.51                            __________________________________________________________________________

Table 6

Systemic Vascular Resistance (SVR), Pulmonary Vascular Resistance (PVR),and Cardiac Output (CO) during NO inhalation with and without Zaprinast.PHTN: pulmonary hypertension. Values are means ± SE.

The arterial plasma cGMP levels during NO inhalation with and withoutZaprinast are shown in FIG. 20. The Zaprinast infusion, by itself, didnot increase plasma cGMP levels. When the Zaprinast infusion wascombined with NO inhalation, however, plasma cGMP concentrations wereincreased at each NO concentration. Nitric oxide inhalationsignificantly increased plasma cGMP concentrations at all NO levelsduring the Zaprinast infusion but only 20 ppm NO caused a significantincrease without Zaprinast. The U46619 infusion alone did notsignificantly change plasma cGMP concentrations.

b. Transpulmonary Difference of Plasma cGMP Concentration During NOInhalation Without and With Zaprinast.

Transpulmonary differences of cGMP concentration in two animals areshown in FIG. 21. The transpulmonary difference of cGMP concentrationwas unaffected by the zaprinast infusion at all levels of NO inhalation.

The maximum pulmonary vasodilating effect of inhaled nitric oxide occurswithin 2 minutes after commencing the inhalation and disappears within2-3 minutes after stopping the inhalation (FIG. 22). Rapid combinationwith hemoglobin in red blood cells inactivates inhaled NO, byrestricting vasodilation to the pulmonary vascular bed (Rimar et al.,Circulation 88:2884-2887, 1993). Although this selectivity is a uniquecharacteristic of inhaled NO, the short duration of action could be adisadvantage because most patients with pulmonary hypertension requirecontinuous therapy. Inhalation of gas mixtures containing highconcentrations of NO and NO₂ causes severe acute lung damage withpulmonary edema and marked methemoglobinemia (Clutton-Brock, Br. J.Anaesth. 39:345-350, 1969). Although there is little evidence for NOtoxicity at low concentrations (<100 ppm) with acute and chronicexposure in rats, little data is available concerning prolonged exposein humans. Since NO is rapidly oxidized into NO₂ in oxygen, the toxiceffects of NO₂ (cytotoxic and immunologic reactions such as type IIpneumocyte hyperplasia and accumulation of fibrin, polymorphonuclearcells and microphages in alveoli) are also of concern, especially duringprolonged exposures. Conceivably, pharmacological agents whichpotentiate and/or prolong the vasodilatory effects of NO might minimizethe risk of NO toxicity during prolonged exposure.

In the present study, it was demonstrated that by using concomitantintravenous administration of a cGMP-specific PDE inhibitor, Zaprinast,the pulmonary vasodilating action of inhaled NO could be prolongedwithout altering its pulmonary selectivity. During the Zaprinastinfusion, the pulmonary vasodilation produced by 4 minutes' NOinhalation persisted 15-30 minutes after the discontinuation of NO (FIG.22). Intermittent inhalation of NO under such conditions could attenuatepulmonary artery hypertension for prolonged period (FIG. 22).

Multiple molecular forms of cyclic nucleotide phosphodiesterase havebeen identified in a number of tissues, including cardiac muscle,vascular smooth muscle, liver, lung, and platelets (Lugnier et al.,Biochem. Pharmacol. 35(10):1743-1751, 1986; Souness et al., Br. J.Pharmacol. 98:725-734, 1989; Silver et al., 150:85-94, 1988; Weishaar etal, Biochem. Pharmacol. 35:787-800, 1986). In mammalian vascular smoothmuscle, three different forms of PDE have been identified: a Ca⁺²/calmodulin-insensitive isoform showing substrate selectivity for cGMP(cGMP PDE, a Ca⁺² /calmodulin-sensitive isoform which hydrolyzes bothcGMP and cAMP (Ca⁺² PDE), and a cAMP-specific isoform (cAMP PDE)(Lugnier et al., Biochem. Pharmacol. 35(10):1743-1751, 1986; Souness etal., Br. J. Pharmacol. 98:725-734, 1989). Zaprinast has been shown todose-dependently increase intracellular cGMP concentrations byselectively inhibiting cGMP PDE (Lugnier et al., Biochem. Pharmacol.35(10):1743-1751, 1986; Souness et al., Br. J. Pharmacol. 98:725-734,1989). Zaprinast-induced relaxation of endothelium-intact rat aorta isgreatly reduced by methylene blue, a guanylate cyclase inhibitor, ordenudation of the aorta (Souness et al., Br. J. Pharmacol. 98:725-734,1989). These results suggest that Zaprinast induced vasorelaxation isdependent on cGMP PDE inhibition and the resultant accumulation of cGMPproduced by basal EDRF/NO release.

Other embodiments of the invention are within the following claims.

What is claimed is:
 1. A method for treating or preventing reversiblepulmonary vasoconstriction in a mammal, which methodcomprisesidentifying a mammal in need of such treatment or prevention,providing a therapeutically-effective amount of gaseous nitric oxide forinhalation by the mammal, and before, during, or immediately after saidgaseous nitric oxide is inhaled by the mammal, introducing into themammal a therapeutically-effective amount of a phosphodiesteraseinhibitor selected from the group consisting of1-cyclopentyl-3-methyl-6-(4-pyridyl)pyrazolo 3,4-d!pyrimidin-4-(5H)-one;(+)-6a,7,8,9,9a,10,11,11a-octahydro-2,5-dimethyl-3Hpentalen(6a,1,4,5)imidazo 2,1-b!purin-4(5H)one;2-phenyl-8-ethoxycycloheptimidazole; and sodium 1-6-chloro-4-(3,4-methylenedioxybenzyl)-aminoquinazolin-2-y!piperidine-4-carboxylatesesquihydrate.
 2. The method of claim 1, wherein said pulmonaryvasoconstriction is acute pulmonary vasoconstriction.
 3. The method ofclaim 1, wherein said mammal has or is at risk of developing a clinicalcondition selected from the group consisting of pneumonia, traumaticinjury, aspiration or inhalation injury, fat embolism in the lung,acidosis, inflammation of the lung, adult respiratory distress syndrome,acute mountain sickness, post cardiac surgery acute pulmonaryhypertension, persistent pulmonary hypertension of the newborn,perinatal aspiration syndrome, hyaline membrane disease, acute pulmonarythromboembolism, acute pulmonary edema, heparin-protamine reactions,sepsis, hypoxia, asthma, and status asthmaticus.
 4. The method of claim1, wherein said pulmonary vasoconstriction is chronic pulmonaryvasoconstriction which has a reversible component.
 5. The method ofclaim 1, wherein said mammal has or is at risk of developing a clinicalcondition selected from the group consisting of chronic pulmonaryhypertension, bronchopulmonary dysplasia, chronic pulmonarythromboembolism, idiopathic pulmonary hypertension, and chronic hypoxia.6. The method of claim 1, wherein said nitric oxide is inhaled in apredetermined concentration range for at least three minutes.
 7. Themethod of claim 1, wherein said gaseous nitric oxide is provided at aconcentration of at least 0.01 ppm.
 8. The method of claim 1, whereinsaid gaseous nitric oxide is provided at a concentration of at least 0.5ppm.
 9. The method of claim 1, wherein said gaseous nitric oxide isprovided at a concentration of at least 5 ppm.
 10. The method of claim1, wherein said inhibitor is introduced into the mammal by an oral,intravenous, intramuscular, subcutaneous, or intraperitoneal route. 11.The method of claim 1, wherein said inhibitor is introduced into themammal by providing an aerosol or dry powder comprising said inhibitorfor inhalation by the mammal.
 12. The method of claim 11, wherein saidinhibitor is inhaled in a gas comprising said gaseous nitric oxide. 13.The method of claim 1, wherein the mammal is a human.
 14. A method fortreating or preventing pulmonary vasoconstriction in a mammal, whichmethod comprisesproviding a therapeutically-effective amount of a nitricoxide-releasing compound to a mammal for inhalation; and before, during,or immediately after said nitric oxide-releasing compound is inhaled bythe mammal, providing a therapeutically-effective amount of aphosphodiesterase inhibitor to the mammal for inhalation, wherein thephosphodiesterase inhibitor is selected from the group consisting of1-cyclopentyl-3-methyl-6-(4-pyridyl)pyrazolo 3,4-d!pyrimidin-4-(5H)-one;(+)-6a,7,8,9,9a,10, 11,11a-octahydro-2,5-dimethyl-3Hpentalen(6a,1,4,5)imidazo 2,1-b!purin-4(5H)-one;2-phenyl-8-ethoxycycloheptimidazole; and sodium 1-6-chloro-4-(3,4-methylenedioxybenzyl)-aminoquinazolin-2-y!piperidine-4-carboxylatesesquihydrate.
 15. A method for treating or preventingbronchoconstriction in a mammal, which method comprisesidentifying amammal in need of such treatment or prevention, providing atherapeutically-effective dose of gaseous nitric oxide for inhalation bythe mammal, and before, during, or immediately after said gaseous nitricoxide is inhaled by the mammal, introducing into the mammal atherapeutically-effective amount of a phosphodiesterase inhibitorselected from the group consisting of1-cyclopentyl-3-methyl-6-(4-pyridyl)pyrazolo 3,4-d!pyrimidin-4-(5H)-one;(+)-6a,7,8,9,9a,10,11,11a-octahydro-2,5-dimethyl-3Hpentalen(6a,1,4,5)imidazo 2,1-b!purin-4(5H)-one;2-phenyl-8-ethoxycycloheptimidazole; and sodium 1-6-chloro-4-(3,4-methylenedioxybenzyl)-aminoquinazolin-2-y!piperidine-4-carboxylatesesquihydrate.
 16. The method of claim 15, wherein the mammal is ahuman.
 17. The method of claim 15, wherein said inhibitor is introducedinto the mammal by an oral, intravenous, intramuscular, subcutaneous, orintraperitoneal route.
 18. The method of claim 15, wherein saidinhibitor is introduced into the mammal by providing an aerosol or drypowder comprising said inhibitor for inhalation by the mammal.
 19. Themethod of claim 18, wherein said aerosol or dry powder is providedsuspended in a gas mixture comprising nitric oxide.
 20. The method ofclaim 15, wherein said bronchoconstriction is associated with asthma.21. A method of improving gas exchange in the lungs of a mammal, saidmethod comprisingidentifying a mammal for whom an improvement in gasexchange within the lungs would be beneficial; providing atherapeutically-effective amount of gaseous nitric oxide to the mammalfor inhalation, and before, during, or immediately after said gaseousnitric oxide is inhaled by the mammal, introducing into the mammal atherapeutically-effective amount of a phosphodiesterase inhibitorselected from the group consisting of1-cyclopentyl-3-methyl-6-(4-pyridyl)pyrazolo 3,4-d!pyrimidin-4-(5H)-one;(+)-6a,7,8,9,9a,10,11,11a-octahydro-2,5-dimethyl-3Hpentalen(6a,1,4,5)imidazo 2,1-b!purin-4(5H)-one;2-phenyl-8-ethoxycycloheptimidazole; and sodium 1-6-chloro-4-(3,4-methylenedioxybenzyl)-aminoquinazolin-2-y!piperidine-4-carboxylatesesquihydrate.
 22. The method of claim 21, wherein the mammal ishypoxic.
 23. The method of claim 21, wherein the mammal is a humansuffering from a lung injury.
 24. A method of delivering aphosphodiesterase inhibitor into the lungs of a mammal, said methodcomprising providing a phosphodiesterase inhibitor suspended in a gascomprising gaseous nitric oxide to a mammal for inhalation, thephosphodiesterase inhibitor being selected from the group consisting of1-cyclopentyl-3-methyl-6-(4-pyridyl)pyrazolo 3,4-d!pyrimidin-4-(5H)-one;(+)-6a,7,8,9,9a,10,11,11a-octahydro-2,5-dimethyl-3Hpentalen(6a,1,4,5)imidazo 2,1-b!purin-4(5H)-one;2-phenyl-8-ethoxycycloheptimidazole; and sodium 1-6-chloro-4-(3,4-methylenedioxybenzyl)-aminoquinazolin-2-y!piperidine-4-carboxylatesesquihydrate.
 25. An inhaler device comprisinga housing defining(a) achamber containing a phosphodiesterase inhibitor selected from the groupconsisting of: 1-cyclopentyl-3-methyl-6-(4-pyridyl)pyrazolo3,4-d!pyrimidin-4-(5H)-one;(+)-6a,7,8,9,9a,10,11,11a-octahydro-2,5-dimethyl-3Hpentalen(6a,1,4,5)imidazo 2,1-b!purin-4(5H)-one;2-phenyl-8-ethoxycycloheptimidazole; and sodium 1-6-chloro-4-(3,4-methylenedioxybenzyl)-aminoquinazolin-2-y!piperidine-4-carboxylatesesquihydrate, and (b) a lumen in communication with said chamber;and avessel containing pressurized gas comprising at least 0.1 ppm nitricoxide, said vessel having a mechanism for controllably releasing saidgas into said chamber, thereby suspending said inhibitor in saidreleased gas; said lumen being configured to route said released gasinto a patient's respiratory system.